Emergency pathology

Author: Mikael Häggström
This article deals with the more time-sensitive matters encountered within pathology, and is written mainly for new pathology trainees.

Memorization-worthy: Information relating to emergent pathology is often not conveniently and timely looked up when needed because of the need for a fast report.

Comprehensiveness

On this resource, the following formatting is used for comprehensiveness:

• Minimal depth
• (Moderate depth)
• ((Comprehensive))

Frozen sections

Even new pathology trainees may end up being the first responders to frozen sections (as well as intraoperative consultations by gross inspection only).

Preparation

Prepare at least the following:

• Finding out which senior to call for help if responding to a frozen section. A fairly new pathology trainee should generally not independently make a report to the referring physician without having at least consulted with a senior, and therefore the diagnostics of frozen section slides is not included in this section. In the meantime, ensure that you have the contact information to relevant seniors and/or attendings, and the means to perform micrography and telepathology if that person may not be able to be physically present within an acceptable time. Also consider calling additional colleagues, even at your own level or below, if there are many specimens coming at once.

Main sites of metastases for some common cancer types. Primary cancers are denoted by "...cancer" and their main metastasis sites are denoted by "...metastases".

• If possible, look up pertinent medical histories of potential cases that may appear as frozen sections or other forms of intraoperative consultations. For example, surgery departments may have schedules of patients for the day that you will cover frozen sections. On such lists, types of surgeries that often come as intraoperative consultations mainly include potentially malignant skin excisions, lung excisions (larger than biopsies), ovarian tumors, and samples from other common metastasis sites (lungs, liver, brain, bone). The most important details to find out are:

• What you may do yourself if you will be alone when you get the specimen, or if you need to wait for particular seniors before doing potentially irreversible steps such as inking and sectioning.
• Previous biopsies. For expected excisions from common metastasis sites, look thoroughly for any past cancer diagnoses. Note the pathologic diagnoses of the biopsies((, as well as the collection dates and accession numbers. Pull out previous slides of the malignancy so that you can compare its appearance with the current case.))
• Tumor sizes where applicable(, and whether the estimation was from imaging or microscopy).

• Make sure you have a working microtome. Switch its blade if you are not certain it has had limited use.
• Prepare at least as many chucks as you think you will need according to any surgery schedule (or at least 3 of preferably various sizes).

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  You can theoretically put a specimen directly on the chuck plate, but to reduce the risk of tearing, put embedding medium on empty chucks

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  Wait until the embedding medium is frozen at the bottom

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  Place the conductor on top of the chuck. You now have a prepared chuck with a smooth surface.

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  Smear some oil on the bottom of the conductor, to avoid it getting stuck to the specimen later.

(If you don't have a conductor, or if the embedding medium completely hardened before you applied it, perform cryotomy of the embedding medium to get a larger and smoother top.)

• Inspect the H&E staining containers and refill if necessary.

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  Example of material needed for H&E staining of frozen sections: Staining fluids, glass slides, pencil to mark slides (the ink of pens will generally be washed off in the fluids), slide dipper, timer, cover slips and immersion oil.

• Ensure that you have frozen section medium, as well as cover slips and mounting fluid.

Performing frozen sectioning

• Inform relevant seniors.
• Look at any requisition form or otherwise check or confirm what answers the requesting clinician wants. When the histopathologic type of tumor is known from before and its distance to the closest margin is relatively far, a gross intraoperative consultation is generally enough to measure that distance.
• Measure the size of the specimen.
• If you are permitted to proceed with the following steps, which are potentially irreversible, still consider whether there are any surprising aspects that requires you to wait until a senior arrives.
• Ink relevant margins
• 'Section the specimen so as to be able to see relevant pathologies, and closest distances to relevant margins.
• Select tissue to be able to answer the clinician's questions.
• Trim samples to make them equally thick, since you will need to section away more from thick pieces before thinner pieces will appear in cryotomy slices.

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  Put specimens on one or more chucks. It's better to put them on a slightly too large chuck than a slightly too small one, but keep the areas of interest within what presumably fits on a microscopy slide.

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  Cover the specimen with embedding medium

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  Apply a conductor (unless it's a thin specimen that needs to stand on its side)

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  Use freeze spray to quicken the freezing if available

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  Breaking off any embedding medium that reaches below the chuck's plate

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  Fasten the chuck on the cryotome. Epithelial surfaces should be oriented vertically, like a picket fence.

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  Generally set the thickness to 5μm, and advance the specimen over the blade while holding the section down to prevent it from folding onto itself

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  Continue until all the tissue of interest is in the section. Leave at least a small part connected to the chuck to avoid folding.

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  Put a glass slide on the tissue (this case is actually borderline too spread out to fit on one slide). It is now ready to be put in staining solutions:

Minimal time in solutions for frozen sections.

Test question Hematoxylin in frozen sectioning You are put on frozen sectioning duty, and a specimen arrives. You inform the attending over the phone, who will require a moment to reach the room, and therefore tells you to prepare two microscopy slides in the meantime. You make sections, but the note that instructs the number of dips or duration in the hematoxylin solution has become unreadable due to a hematoxylin stain. You don't have any phone signal in the frozen section room, so you don't have time to look it up. To what extent will you put the slide in the hematoxylin solution? A. 1 minute and 30 seconds B. 1 dip C. 10 seconds D. 5 dips Answer Explanation This is generally the minimal time required for proper hematoxylin staining during frozen sections. Yet, if you know the solution is not expired or diluted, and you are really in a rush, then you can possibly have it acceptably stained in 1 minute in hematoxylin.

Troubleshooting

Problem	Cause	Solution
Tissue cracks off in cryotomy	Block too cold	Repair by warming slightly and adding embedding medium
Thick and thin slices	Inconsistent clamping Dull blade Incorrect blade angle	Ensure block holder and blade holder are tight Change blade Correct knife angle
Wrinkled, compressed or puckered sections	Dirty or dull blade Tissue too warm	Clean blade Move or replace blade Cut tissue at colder temperature
Tissue advances but does not cut	Inconsistent clamping	Ensure blade holder is tight
Sections roll up	Anti-roll plate is not adjucted correctly	Readjust anti-roll plate
Sections thaw on the blade	Blade is too warm	Cool the blade
Sections stick to the anti-roll plate or blade	Static in the cryostat	Apply anti-static spray or anti-static gun
Sections split vertically	Dirt or nick on blade	Clean, move or replace blade
Sections have fine cracks parallel to the blade edge	Too cold temperature	Warm the tissue
Sections show signs of vibrations	Specimen too loose Incorrect blade angle	Tighten chuck to the holder Change the blade angle
Stain is dull and muddy	Drying artifact	Put sections on glass slides immediately in fixative

Thawing

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  When no more frozen sections are needed for a block, do not use water directly on the sample, as it may get flushed away.

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  Rather, apply hot water to the stem of the chuck, while slightly tilting your grip of the top to apply pressure towards making the block slide off the chuck. As soon as you feel it starting to move, move away from the sink and finish sliding the block off the chuck.

Reporting of intra-operative consults

• Call the requesting clinician
• Verify that you are talking to that person, or someone who can immediately convey the diagnosis to that person. You may request to talk directly to the surgeon if a conversation is warranted.
• Tell the patient identity (with at least two different identifiers, such as name and date of birth)
• (Ask for a readback of your diagnosis. This is particularly important in cases with multiple specimens, and when it needs to be stated that a more specific diagnosis is deferred until standard slides have been evaluated.)
• State your diagnosis succinctly. If necessary in cases with limited diagnostic tissue, you may state that additional tissue is required for a more certain report.
• (Make sure the clinician reads the diagnosis back to you.)

Example:

Hello, this is from pathology. I have a frozen section result for Dr. (name of the clinician) This is Dr. ____, and I have a diagnosis on (patient name and date of birth)(, if you could please read back to me after,) the diagnosis is (diagnosis)

Frozen sectioning of suspected malignant skin excisions

While most frozen sections can be predicted from schedules of the operating room and thereby be looked up beforehand, suspected malignant skin excisions often come from outside the main surgery department, even more indicating memorization of how to handle them.

Tissue selection

Tissue selection from suspected malignant skin lesions, by lesion size:

<4 mm	4 - 8 mm	9 - 15 mm

In table above, each top image shows recommended lines for cutting out slices to be submitted for further processing. Bottom image shows which side of the slice that should be put to microtomy. Dashed lines here mean that either side could be used. Further information: Gross processing of skin excisions

Donor kidney biopsy

Gross processing

Prepare a separate slide for right versus left kidney if applicable.

Microscopic evaluation

Generally evaluate as per local protocol, which generally includes the following parameters, given individually for right and left kidney:

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  - Number of glomeruli with global sclerosis
  - Total number of glomeruli.
  - Percentage of glomeruli with global sclerosis (number of glomeruli with global sclerosis divided by total number of glomeruli).

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  Tubular atrophy/tubular interstitial fibrosis, as percentage of tubules. Features of tubular atrophy are shown (of which casts is often not present).

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  Arteriosclerosis seen as intimal thickening with an onion skin-like appearance. Arteriosclerosis can be graded as mild, moderate or severe.

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  Arteriolar hyalinosis, graded as mild, moderate or severe.

Other frozen sections

Although these are generally given on schedules of the operating room, any pathologist may end up suddenly covering for another one, and subsequently be presented with the frozen section case without having had the time to look it up beforehand.

In malignant or suspected malignant cases, surgical margins are generally more important than main tumor diagnosis.

• In lung wedge resection and lobectomy, sample the surgical margin that is presumably closest to a tumor for frozen sectioning. Further information: Lung wedge resection and lobectomy
• In an intestine with tumor, it is generally enough to open the intestine (avoiding cutting through a tumor unless it is corcumferantial, and grossly measuring the distances to the proximal and distal surgical margins, as well as the distances to the radial or mesenteric surgical margins. Further information: Intestine with tumor
• For a donor kidney biopsy, generally evaluate as per local protocol. Further information: Donor kidney biopsy

However, for myocarditis requests (see the heart article), generally have the cardiac biopsy sent in formalin for regular H&E processing (even on weekends) rather than perform frozen sectioning, since good quality slides is the priority.

See also

• Common on-call topics in clinical pathology

• Low blood glucose
• Suspected blasts on peripheral blood smear

Main page

• Main page of Patholines