Easy Ways To Improve Your Histopath Results, Part 1

In the first part of a four-part series, pathologist Ken Mero, DVM, Ph.D. sheds some light on histopathology samples.

Giant chest mass: This chest wall fibrosarcoma on a 12-year-old male pit bull mix will need to be cut into pieces in order to be fixed properly by formalin.

Dr. Phil Zeltman

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Fine-needle aspirate or biopsy? Slice or dice? Vials or bags? We asked pathologist Ken Mero, DVM, Ph.D. (VetPath Services, Stone Ridge, N.Y.) some important questions about histopathology samples.

We often remove large masses that won’t fit in a regular formalin jar, or half a mandible that we can’t cut into. How should we handle these samples?
Here are a few options:

• Use a larger jar, because you can’t fold a mandible
• Allow fixation for several days at the clinic in a large container, later transferring to a smaller container with a smaller quantity of formalin for transport
• Select one or more portions of a large mass for transport in a reasonably sized formalin jar or possibly several jars
• Pack the specimen in formalin-soaked gauze sponges or towels in a suitable, reasonably sized container. The sample can then be transferred into an adequate volume of formalin at the laboratory.

The main detriment to the latter is limited penetration of formalin from the specimen surface into its interior and therefore the potential for central autolysis and impaired histologic interpretation.

If you submit a large specimen in plastic bags rather than jars, secure double or triple Zip-loc bagging is recommended.

Is cutting into a large mass acceptable to help formalin penetration?

If you must, then a single incision is preferable to “bread-loafing,” which may impair the pathologist’s selection of optimal sections.

What can we do to help our pathologist colleagues?

We really like a good clinical history. Many diagnoses are not a slam-dunk simple conclusion based on very characteristic microscopic features, but are a combination of clinical signs, history and microscopic findings. Some pertinent historical features might include known past injuries or illnesses. If a tumor was previously excised, we’d like to know which type and see the biopsy report.

Patient signalment (age, sex and breed) is also important. Not only for the diagnosis, but also to give a prognosis and perhaps influence a treatment recommendation. With regard to neoplasms, helpful information includes lesion size, gross appearance, specific anatomical location, duration and clinical behavior such as growth rate or recent changes in the latter.

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You can also help your pathologist with specimen orientation by drawing a picture and/or identifying with sutures, staples or ink.

Last but not least, adequate labeling of specimen jars, fluid containers or slides will prevent confusion and mix-ups at the laboratory.

Does the type of biopsy matter to you?

Sure, we would like to know the method of biopsy. Is it incisional, excisional, complete (with estimated dimension of margins)? Is it a needle core, Tru-cut, punch, endoscopic, wedge biopsy?

With regard to wedge biopsies, there is potential confusion regarding a tissue wedge containing the whole mass or an intralesional or incisional wedge, representing a portion of a larger mass. Therefore, when indicating a wedge biopsy, further definition as an “excisional wedge” or “incisional  wedge” is important.

In addition, if you submit an excisional biopsy, it is helpful to distinguish among a sharp scalpel excision, a blunt dissection or a mass which “peeled” or “shelled” out of a tissue, organ or fascial plane.

It is also helpful to know if a lesion had a pre-biopsy cytologic evaluation and if so, what the results were.

If margin evaluation is requested on a large specimen, submission of the biopsy in its entirety is optimal, even if a large specimen has to be held at the clinic in an adequate volume of formalin for adequate fixation before shipment in a smaller container to the laboratory.

What is the ideal size for the formalin container?

Too much formalin is better than too little. As a general rule, formalin penetrates tissue at a rate of approximately 1 mm/hour, but can only penetrate a maximum of 5 mm into solid tissues (exceptions are cystic structures, eyes, brain, etc.). Therefore, the ideal sample biopsy size is about 1 cm in every direction.

There are very standard guidelines regarding the ideal tissue:formalin volume ratio. Ideally, the formalin volume should be 15 to 20 times the volume of the tissue. If this is not practical to transport to the lab, then fixation in a large jar for a few days at the clinic is additional time well spent since the optimally fixed specimen will generate better histologic preparations.

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Most small to moderate-size specimens are adequately fixed during transit to the laboratory in the standard-sized jars provided. The detriment to too much tissue with too little formalin is poor fixation, autolysis of the lesional center and the necessity for additional fixation at the lab, which will delay processing and may impair diagnostic accuracy.

When should we perform a fine-needle aspirate (FNA) vs. a biopsy?

Aspiration has many possibilities, ranging from providing a “general indication” to a definitive cytologic diagnosis. It can also provide directions for the next course of action, which is often a biopsy. Aspiration is most likely to be diagnostic for body fluids, such as body cavity effusions, CSF, joint fluid or intraocular fluid.

Some masses also are good subjects for FNA to distinguish between inflammatory and neoplastic processes. Round cell tumors (mast cell, histiocytic, plasmacytic, lymphoid) are good candidates for FNAs.

Remember that tumors of round cell and epithelial origin generally exfoliate reasonably well, whereas those of mesenchymal origin often do not, because of the tighter cellular cohesiveness of mesenchymal cells.

When are the benefits of a FNA questionable?

Some situations where aspiration diagnostic accuracy is questionable include lymph node aspirates, because there may be significant cytologic overlap between advanced lymphoid hyperplasia and early emerging lymphoid neoplasia. This distinction is often histologic.

Yet with advanced lymphoblastic lymphosarcoma with lymph node effacement, FNA may be enough to reach a diagnosis.

FNAs of hemorrhagic or parenchymal lesions may contain blood elements that can lead to misleading or disappointing results.

Also, there is often difficulty in the cytologic distinction of benign vs. malignant tumors of glandular epithelial origin (most notably mammary) since benign mammary tumors may be cytologically highly atypical while malignant tumors may not be bizarre or mitotically active. Both benign and malignant plasma cell tumors may be similarly cytologically atypical or bizarre.

Again, FNA can be a preliminary step toward biopsy.

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How frequently are FNA and biopsy accurate?

This depends on the nature of the fluid or tissue aspirated and the assumption that the sample submitted is representative. In many cases, FNA may indicate a neoplasm of round cell, epithelial or mesenchymal origin, but it may not be a reliable verification of benign or malignant status, particularly a lower grade malignancy.

Biopsy may be more accurate because mast cell tumor (MCT) grading cannot be done cytologically, only histologically. In addition, only a biopsy allows assessing surgical margins.

Be aware that the grading of canine cutaneous MCTs actually involves not only the status of mast cell differentiation, but also tumor behavior at its periphery (well-defined and expansile, or ill-defined and invasive).

The behavior of MCTs does not always correlate directly with the state of cellular differentiation. Also, mast cell tumor grading is restricted to cutaneous lesions of dogs. The classification does not apply to cats or visceral MCT.

This is the first part of a four-part series. Click here to read Part 2.

Dr. Zeltzman is a mobile, board-certified surgeon near Allentown, Pa.


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