Skin excision is both a diagnostic and a treatment procedure. In most cases, it follows shave and punch biopsies. Although most excisional biopsies have elliptical shape, some of them can be oblong or irregular form. Skin excision presents epidermis, dermis and subcutaneous tissue. Some accessory structures of the skin (glands, hair follicule, and erector pilli muscle) might be involved as well.
In contrast to incisional biopsy that has only a diagnostic value, the main skin excision biopsy’s goal is the complete removal of the lesion. Incisional biopsy is used rarely in practice. An incisional or even full-thickness punch biopsy might be used in a melanoma case to obtain maximum depth of penetration from a nodular area of the tumor.
Elliptical (wedge) excisional or incisional biopsies are preferred when the disease process involves the deep dermis or subcutis. Superficial fascia can be obtained reliably only with this technique.
Excisional biopsy is recommended in all melanocytic lesions. Neither punch, nor shave biopsies can substitute excisional biopsy because they cannot give all necessary information.
Inking is obligatory to give credible information about the margins of excision.
One color inking is sufficient in skin excision without orientation. Two colors inking in the ellipse excision with orientation is the most efficient, because the tips are clearly distinguishable.
Four color inking is reasonable in oval/semi oval excision with orientation.
It is understandable that gross dictation should reflect orientation after inking. Usually clockwise marks are appropriate. When dictation with ink and suture o’clock designation is used, the dictation should include not only o’clock numbers but designate direction “from/ to” (from 12 to 3, from 3 to 6, from 6 to 9, from 9 to 12 when the sections are placed in the cassettes)
Large flaps of excised skin with malignancy, which have been described in old books, are now rare due to earlier diagnosis and treatment. These specimens require subdivision and labeling. Each subdivision should be inked and examined like a separate biopsy with mapping that will present the entire specimen.
The methodology of inking is presented in Common Techniques in Skin Grossing section.
Although application of each ink as a separate procedure is optimal, it is not used in practice as time consuming and unreasonable. However, careful blotting and air drying is very important to maintain the ink on the specimen and precisely at the place it intended to be.
The mode of sections depends on size of the specimen, type of the excision (ellipse, round, irregular, flap), and diagnostic goals (lesion, margins).
The working classification by size is:
small 2 cm; regular 2- 5cm; large 5-10 cm; and extra large (more than 10 cm).
The evolution of the skin ellipse’s size submission in Toto shifted from 2.5-3.0 cm to 5 cm. The policy regarding large specimens depends on the institution’s protocols. For example, in a wide re-excision of previously excised malignant melanoma, after the sampling of the scar, the surrounding pigmented areas, and the margins, the remaining tissue needs to be stored, if the entire specimen is not submitted. It is more rational to keep the remaining specimen in properly labeled blocks and slides than wet in containers as it was often recommended before.
The table presents suggestions for inking and sectioning
|Type of excision||Ink||Optimal sections|
|Ellipse without orientation||One color||2 or more|
|Oval/semi oval without orientation||One color||2 or more|
|Ellipse with orientation||Two/four colors||4 or more|
with orientation Four colors4 or more.
A typical skin excision with orientation specimen. Below is the sequence of grossing actions.
Drying with a blotting paper.
Applying a mordand to fix the ink (Bouin’s solution, but can be 5% acetic acid alone)
Inking in two colors.
Drying after the mordand.
The tips are cut the first
Vizualization of the lesion is the main priority during skin grossing section.
The lesion should be presented completely without ambiguity during embedding.
The last section between the forceps. Each tip is placed in a separate cassette.
The rest in sequence clockwise
Direction of the section
Direction of section in the skin excision sample depends on the type of surgical procedure and the goal of evaluation (surgical margins, neoplasm’s characteristics). There are three types:
a/ regular- vertical as much as possible perpendicular;
b/ Mohs surgery- horizontal, oblique to parallel;
c/ special studies- horizontal as much as possible parallel.
These materials discuss only regular dermatopathology procedure. Mohs surgery- “microscopic-controlled excision of cutaneous tumors” has developed in a special subdivision with specific methodology that requires separate description. It is well presented in manuals and other materials.
Vertical sections have three variants: transversely, perimeter, longitudinal.
a/ Transversely through the skin sample including the area of diagnostic interest (bread-loaf sections) – the most ubiquitous (diagram 1).
Bread-loaf section assumes serial sectioning along the short axis. The sections are perpendicular to the specimen’s margins and margins of interest. This is particularly beneficial in melanoncytic lesions. Perpendicular bread- loaf sectioning contributes to three dimensional image which is important in melanoma diagnosis. It is the standard to submit the entire cross-sectioned ellipse, with the exception of extremely large specimens, where representative perpendicular margins can be taken around the clock face, and the biopsy site scar can be submitted in total (see diagram 6 below).
b/ Perimeter, or peripheral section, or en face, the best for margins evaluation (diagrams 2, 3, ).
En face sections provide two dimensional approaches which is sufficient in skin cancer diagnosis and margins. To submit en face margin, the cut surface (non-inked) is placed face down on the cassette. This surface will approach the microtome blade. This circumstance allows the pathologist request deeper levels of the block to reach the true margin.
Although this mode of sectioning is the best to visualize margins, it is better on the paper diagram than on the grossing table. The specimen is difficult to immobilize during sectioning. The technique provides slices uneven thickness. The sections are technically difficult to orient during embedding. This method can be used predominately in large slaps of the skin with a wide rim between the margin and the area of interest.
As a general rule, in melanoma cases a 0.5 cm margin required for superficial lesions and 1.0 cm margin for deep lesions. These considerations should determine the en face methodology of sectioning.
There might be variants of sectioning and submission when only parts of the lesion are submitted leaving unsectioned parts free of the lesion (diagram 4). However, this mode of sectioning brings some uncertainties which are better to avoid.
c/ Longitudinal through the skin sample and the area of diagnostic interest (bread-loaf-cross technique)- rarely used in large tumors (diagram 5).
Longitudinal single section obviously does not examine the surgical margins. It is closer to an incisional biopsy. There is no justifiable reason to use this method under conditions of advancements in modern histology processing techniques and equipment.
Depending on the institution’s protocols or the pathologist’s preference, for large or extra large (greater than 15 cm) excisions, especially with a scar after wide melanoma excision, only radial margins are taken, while blocking out the scar or lesion and submitting them entirely after serial sectioning. In this situation, radial margins are taken the first. Then more or less a rectangular block of the area of interest is taken out, and then the block is serially sectioned. However, it is easier to do this at the paper diagram than on the grossing table. The requirement is to leave the specimen’s integrity intact as much as possible to allow the pathologist to return to the specimen if required (diagram 6).
There can be different combinations of submission when the central part is submitted entirely, but the remaining central area is submitted as representative 50 to 80 %%.
Old books, Rosai for example, present combination of these variants, especially in malignant tumors. The most common practice prefers bread-loaf method (Lester).
“Square” procedure, or the “polygonal method”, is one of excision techniques of treatment for Lentigo Maligna. It is called also “slow Mohs” (staged, margin-controlled excision with rush permanent sections). The standard excision margins of 0.5 cm are often inadequate due to the subclinical extension that can occur. “Slow Mohs” is the staged excision of peripheral and deep margins with rush permanent sections, followed by repair of the resulting wound. Full-thickness skin strips 2mm in width are harvested 0.5cm from the LM. The outside circumferential margin of the strips is marked with permanent ink, and the pathology lab is instructed to section the strips tangentially along this inked margin. Strips are divided so that they are approximately 2.5cm long and will fit inside a standard paraffin embedding cassette. Various mapping methods to document the location of the harvested skin strips such as marking suture, permanent marking ink, or incisional nicks in the skin are used.
Usually the specimen is sent to the laboratory accompanied with a diagram, even a photograph. In Stage 1 frame procedure with punch biopsy from central pigmented region.
The “frames” are cut off (shaved) by perpendicular sections and placed each in a separate cassette with preliminary inking, for example in green the false inner margin, in red outer margin).
The frames (A, B, C, D, etc. or superior, posterior, inferior, anterior, or 1, 2 3, 4, etc.) should be embedded with inner margin down so that the sections can reach free, if any, of tumor margin. The central portion is submitted depending on orientation and the necessity to examine representative or entirely ( diagram 7).
The tips are essentially en face margins. If a neoplasm is present in the tips but does not extend to inked surface, margins could still be positive. One would need to level through the block at that point to be sure, but anyway this is a bad sign.
There is no unanimity in approaches to grossing tips in ellipse excision. Some dermatopathologists do not pay special attention to them at all in excisions larger than 3 cm in length.
The most conventional type of tips presentation is transverse cut (diagram 8).
It is technically most preferable for grossing and embedding. However, theoretically remains some space for uncertainty between the base of triangle cut and the upper angle. There is always an opportunity to cut deeper if the tip’s margin does not satisfy the pathologist.
Different approaches to tips presentation try to solve this uncertainty, for example, the tips are bisected and embedded at the surface of the cut (diagram 9)
However, this does not solve the problem of complete presentation because the peripheral parts are excluded from the pathologist’s view. In small specimens the bisected fragments is difficult to embed properly.
Another way of tips presentation as cutting in three fragments with embedding the central part is also questionable, besides it is unclear how to embed the minuscule remaining fragments (diagram 10).
The sharper is the tip’s angel , the most appropriate it is to put it in an orientation pad without any attempts to cut it. Otherwise, the tip is curling and embedding might be confused.
In rare occasions, the surgeon may notch the ends of the ellipse for easier healing of the wound. These serrated parts should be considered as tips. Sometimes, the specimen includes the triangular fragments (“dog ears”) which also are technically tips.
The grossing manuals usually suggest starting with a cut through the most prominent area, especially in pigmented lesion (“the thickest or darkest portion” –Lester). However, it is only formally correct. The entire lesion should be tailored in the way that every section has complete presentation without ambiguity during embedding. For example, the left section (picture below) can be embedded in the way that pigmented area would be at the opposite side of microtome section. Of course, an ink mark to inform how to embed can be used in this situation, but this is not the right way to cut. The section ought to be as much as possible unambiguous for embedding.
Preliminary, the lesion should be cut from both sides. The lesion should then be serially sectioned keeping in mind the necessity to present the most prominent grossly pathology while following the modern more strict requirement for uniformity of the section’s thickness due to micro wave assisted processing.
The optimal mode how to cut
Both immobilization and visualization are keys in any meaningful section in any biopsy, especially in skin excision. The following illustration presents the optimal mode of sectioning.
Sectioning against a hard flat board would be the best as far as immobilization is concerned. For example, the sample below is placed facing epidermis down. Such position might enable a strong perpendicular section by preserving the vertical stratification of the skin.
However, such blind sectioning is acceptable only if any diagnostically valuable part of the specimen is not involved. The option of the ellipse placement on the side allows the grossing person to visualize the section while maintaining a satisfactory immobilization against the hard flat surface of the grossing board.
The sample, which is placed on the board with subcutaneous tissue down, is inevitably less stable than squeezed between forceps on the back side. The necessity to make a decisive last section of the skin sample requires holding between the forceps’ brunches unless the fragment is completely diagnostically unremarkable.
In Breslow measurements, it is crucial to make perpendicular to the epithelial surface sections because a slight variation away from perpendicularity (22.5 to 45.0) can increase tumor thickness of considerable importance.
Placement in the cassettes
The placement of the section in the cassettes depends on the type of specimen (orientation), protocols of the institution, and personal preferences of the pathologists. For example, Lester manual recommends putting the most diagnostically significant section in the first cassette and the tips in the last. However, for the sake of standardization and the logic of section, and prevention any confusion, in my view, the most rational is following the clock wise description while placing the section immediately in the prepared marked cassettes, although the tips can be placed the first also. If the specimen is inked in one color, the tips can be placed in one cassette.
In dictation with ink and suture o’clock designation is used with dictation not only o’clock numbers but from/ to (from 12 to 3, from 3 to 6, from 6 to 9, from 9 to 12 when placed in the cassettes).
In the “bread-loaf” method, the ellipse is transversely sectioned at approximately 3 mm intervals. If the cut surface is marked with ink and each tissue slice is embedded in a separate cassette, it is easy to decide which block to recut if additional sections are needed.
It is unreasonable to place more than two sections in the cassette to prevent unevenness during embedding.
It makes sense to place tips in the incision in a foam pad, if confusion might occur how to place them in the embedding mold,. Such confusing can definitely occur if the tips are very thin.
Cyst in skin excision
Sebaceous cyst should be wrapped otherwise the content might clog the processor.
The main goal of the examination is to find hair in subcutis and to determine that excision
is satisfactory complete. It is necessary to submit the most representative (with possible hair or pus. The specimen should be ink to provide information about completeness of excision just in case if required.
Lip excision is described in detail in Lester’s manual. The diagrams are informative.
On the other hand, the Lester’s manual refrains from discussing nails grossing although these specimens are not rare in dermatopathology not only for fungal infection.
Two areas of diagnostic interest: clipping the nail for fungal infection and the nail matrix, if presented, for melanoma.
Independently of the purpose, any decalcification is not advisable because they are compound from insoluble keratin filaments that doesn’t have calcium. However, if the nail is attached to bone, it is not a waste of time to put in the decalcification solution (RDO type) because you have to get rid of bone components.
Here is the most universal method of finger/toe nails processing. After fixation, rinse from formalin. Depending on the amount of attached soft tissue, the nail is placed in 5% aqueous sodium chloride (plenty of soft tissue) or 10% ammonia water for alkaline hydrolysis (only nails). Then rinse off the soapy water when the nail becomes pliable(not too vigorously because attachment of fungi might be washed away). Rinsing does not affect anything adversely, it only eliminate the alkali, as well as everything removed from the nails. While sampling, cut uneven sections. The thickest section (usually proximal and the most diagnostically important) should be shorter (0.15cm) than distal (up to 0.3 cm) that provides even processing. This is especially important if melanoma is suspected.
Some trick from practice. “Nair” (hair removal cream) or other depilatory cream (Neet, for example) can be used when completely applied (sometimes overnight and a litttle warm up. Apply completely covered Wipe off- spray with Cytocool and cut. Sometimes requires overnight or a little to warm up. 5% Tween 80 solution for several hours or overnight. 4-6% Trichloracetic acid softens the nail and unlike the traditional alkali solutions does not contribute to lifting or curling of the section on the slide during staining.
Skin excisions are the most complicated skin specimens. However, if the rules are followed, the result is much more certain than in shave and punch biopsies. In skin excision, the grossing person’s role is paramount, unlike in shave and punch biopsies where the role of the histotechnologist is more important, especially at the embedding stage of processing.