Skin Punch Biopsy

Grossing a skin punch biopsy is simpler than a shave biopsy. In a punch biopsy, a plug of skin 2 to 12 mm in diameter is removed using a cylindrical instrument with a sharp cutting edge. The punch biopsy tool is best to obtain the epidermis, dermis, and minimal amount of subcutis. Every punch biopsy gross description should include a characterization of the lesion/pigmented area surface with its location (centrally/eccentrically).

Clinical indications for punch biopsy:

Pigmented lesions

Inflammatory disease/panniculitis


LlDegenerative disease


Painful sensory neuropathy

Toxic Epidermal Necrolysis (TEN)

From diagnostic approach, inking is reasonable only to determine margins although in most cases the clinician does not ask for them assuming that punch biopsy is only diagnostic procedure that is right only in principle. In general, small punch biopsies are unreasonable to ink because it only creates difficulties for embedding. Inking is unwise in cases when vasculitis or alopecia is suspected. Inking is obligatory if a suture tag orientation were accompanied which means that clinician is interested in margins.

To cut or not to cut?

No any cut is reasonable if

the skin surface is less than 3 mm or even 4 mm that can be bisected prior to embedding;

In lesions 1mm or less, the cut would destroy the integrity of the subcutaneous tissue, especially if the clinical diagnosis is vasculitis or alopecia. Thin or eccentric subcutis is better not to cut or leave for a decision during embedding when the subcutaneous tissue is more manageable for a cut.

The table presents approximate suggestion for punch biopsies inking and cutting.


Size in cm Ink Section How
0.2 and less no no
0.3 no/yes no/yes? no/bisection
0.4 -0.5 yes yes bisection
0.6-0.7 yes yes bisection
0.8-0.9 yes yes trisection
1.0 -1.1 yes yes trisection
With orientation  two colors yes trisection

Some illustrations


In a punch biopsy 0.2 cm, a green pin for embedding orientation might be reasonable. The specimen should be embedded intact. Any gross tangential cut when the epidermis is sectioned through can damage the histological skin stratification creating difficulties for the pathologist, like oblique cuts of vessels can give a false impression of the thickness of the wall.

Some suggestions of the cut directions in bisected punch biopsies

The 0.3 cm punch biopsy should be bisected through pigmented area. The suggested direction of the cut displays the location on the margin and leaves enough space for re-cuts. In ideal situation of an experienced histotechnologist, punch biopsies of 0.3 – 0.4 cm is reasonable to bisect before embedding to avoid crush artifact of incomplete fixation (hardening) that might prevent difficulty of specimen orientation during embedding.

This   punch skin biopsy is bisected through the pigmented area to inform about the margins of the pigmented area even if the clinician haven’t ask for them.

The specimen is bisected

Some variants of bisection which emphasis on presenting the pigmented area in the most informative way, including it closeness to the margins.

Punch biopsies ≥8 mm require one color ink, trisection, and placement in two cassettes.

The tag determines two color inking (black and green). The sections of different colors placed in different cassettes with the section with the tag are placed in the third cassette. The disposition of the sections is mentioned in the gross description.

In a more complicated case, the specimens were inked in four colors according to the instructions in the requisition form. The central part was inked in two colors (black and green).

Punch excision is also frequent description of the specimen even if the measurements are as 0.7x07x03 cm. In this situation the surgeon presumes the margin’s check. The specimen should be inked, trisected, and placed in two cassettes.

In rare occasions, if the excisional biopsy were not possible to carry out, a full-thickness punch biopsy can be taken as kind of incisional from nodular area of melanoma with a part of normal skin to determine maximum depth of tumor penetration. However, the excisional biopsy is optimal for step-section analysis and determination of the maximum depth of penetration to determine extent of surgical margin and possibility of regional node involvement.

  Sometimes no shave, not a real punch biopsy is sent to the laboratory. The principle of grossing is similar to the punch excision biopsy.


 Alopecia diagnosis (androgenic or a different origin) requires as a rule a punch biopsy. The principle of the pathologist’s diagnosis is in evaluation of hair follicles per square millimeter, actually counting them on the section parallel to epidermis Most punch biopsies do not excide 4 mm in diameter. Dr J.T. Headington’s transverse microscopic scalp anatomy procedure is used. Depending on institution, there are two protocols: the specimen is submitted in toto (punch biopsy ≤ 4) for processing and all transverse section manipulation is done during embedding (the embedding person is notified by marks on the cassette or tags inside) or the gross section is made on the grossing table. In any event, the specimen is sectioned transversely for embedding the section’ cut surface down in the embedding mold. Some pathologists require multiple (usually four) levels of horizontal sections to the skin surface from epidermis to the subcutaneous adipose tissue. If thee biopsy is ≥ 4 mm the specimen is bisected vertically. One is submitted for routine H&E the second for alopecia protocol.

Some dermatologists send two alopecia punch biopsies: one for transverse section, another for  vertical section. This makes sense keeping in mind that there is plenty of space on a bold head for taking a biopsy. By the way, in CPT coding this case would be 88305×2.

   Skin specimens for Direct Immunofluorescence (DIF) usually arrive in Michel’s solution ( buffered ammonium sulfate. The formalin fixed specimens cannot be used for immunofluorescence studies. The specimen is washed in buffer solution and cut in the cryostat after embedding in OCT. The sections are stained with immunofluorescence reagents.


Usually in the case of suspected epidermolysis, the specimen goes for frozen section and submitted entirely when any orientation is almost impossible. Any inking is unreasonable because it would interfere with staining.

Do not place skin biopsies in saline solution. An artifact, consisting of hydropic degeneration of the basal cells and subdermal bulla formation occur in skin-punch biopsy specimens Inoshita and Youngberg (Am J Clin Pathol 80(2): 206-9, 1983) noted similarities with epidermolyis bulloza simplex or lupus erythematosus.

Painful sensory neuropathy.

3mm punch biopsy is taken for evaluation of density of intraepidermal nerve fibers (IENF) that is reduced in painful sensory neuropathy. The specimen requires 24 hours fixation in 2 % paraformaldehyde, actually in Paraformaldehyde/Lysine/Periodate-PLP fixative. The procedure requires special protocol of cryosections after embedding in sucrose, but at the grossing level of processing, it is necessary to provide adequate fixation. The principles of grossing are the same in Small Fiber Neuropathy (SFN) tests as such as Epidermal Nerve Fiber Density (ENFD) and Sweat Gland Nerve Fiber Density (SGNFD).

Punch biopsies sometimes are difficult for histotech due to tissue heterogeneity (epidermis, dermis, and subcutaneous tissue with a tail of fat tissue on the relatively “small proximity”). This circumstances determine special attention to fixation with unequal formalin penetration, obligatory charged slides, and not the last skilled microtomy with the right blade approach from firmer to soft areas (stratum should face the blade.)

As a general conclusion, the section before embedding in punch biopsies makes sense if the technician is trained in understanding grossing pathology issues and has time to do this, usually not, in the hustle of the processing. Very often paraffin obscures the borders of the pigmented area or the lesion. It seems that the best way is to give the embedding person a finished grossing processing sample because the grossing person has more information about the specimen and means to present the most diagnostically valuable parts of the specimen to the pathologist’s attention.




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