The modern biopsies processing includes many ancillary studies which require different modes of preservation. Fixation is only one though the most ubiquitous technique of preservation. As a variant of preservation, fixation has a dual role. Besides its main purpose of preserving cellular structures, it also ensures the co called hardening of the specimen. In skin biopsies, when differences between specimens by size and consistency are the most prominent, variations in fixation have practical significance. Hardening is an old term that stems from pre-formalin era of alcohol fixation. Actually, fixation provides immobilization of the tissues components by preventing them from moving during cutting procedure that is especially important in skin specimens.
The table summarizes most frequent skin preservations methods although this table is incomplete due to the variety of studies and reagents.
|Type of preservation||Reagent/s||Purpose||Special conditions|
|Fixatives||Formalin, glutaraldehyde, Bouin’s solution, Zink formalin, UMFIX, Prefer, ExCellPlus, etc.||Preserve cellular structure, “hardening” for grossing sampling||Minimal and maximal time of exposure, monitoring|
|Transport medium||Michel’s (Zeus) solution||Preserve for immunofluorescence||Limited time for exposure (2-5 days)|
|Transport isotonic liquid||Normal saline (0.85% NaCl)||Prevent from drying||Short term of exposure due to maceration and autolysis|
An artifact consisting of hydropic degeneration of the basal cells and subdermal bulla formation can occur in skin-punch biopsy specimens if they were kept in saline solution for a prolonged time. This artifact can simulate epidermolyis bulloza simplex or even lupus erytematosus.
The “gold standard” of 20:1 ratio of formalin and tissue is unwarranted. More, studies show that the ratio can be even less than 5:1, especially if modern tissue processors are used that is almost always the case.
In skin biopsies when differences between specimens by size and consistency are the most prominent, variations in fixation have practical significance. Formalin is a slow non coagulant fixative. Although the speed of fixation can be accelerated by higher temperature, vacuum and pressure processors, as well as by microwave assisted processing, the classic notion that 10% buffered formalin penetrates about 1 mm/hr remains as a guiding point. When a “rush” specimen arrives in formalin, it does not mean that the specimen is completely fixed. Change in color of blood can be accepted as an approximate end point of formalin fixation, sufficient for grossing purposes. This is important in skin biopsies which arrive at different stage of fixation. When the red-pink areas of skin biopsy removal turn tan-brown (formation acid formalin hematin), the specimen can be considered ready for tissue processor although the fixation is continued in the dehydrating alcohols.
PROCEDURES in GROSSING SKIN BIOPSIES
Sampling, or cut-in, is the main part of skin biopsy grossing. This procedure requires following appropriate grossing techniques. Biopsies have little space for errors in grossing manipulations. Every action should be precise and decisive.
Two general requirements to skin biopsy grossing technique: representative and informative.
Representative, or adequate – the sample should present the most diagnostically valuable parts of the biopsy specimen for the pathologist’s review.
Informative, or definite – the sample should have clear and unambiguous instructions for embedding and microtomy.
Completeness of submission, the third biopsy requirement, cannot be applied to all skin biopsies due the size of the specimen or a diagnostic impracticality; however the lesion and the margins should be presented as much as reasonably possible.
The representative and informative components are inseparable. Ink and knife are means for nonverbal communication between the grossing person and the pathologist, as well as the histotech.
If the ink does not carry any message, it does not make any sense to ink. In a thin skin specimen, insoluble ink fragments can diffuse from the area of inking obscuring the pathologist’s vision. In all questionable situations it is reasonable to abstain from inking.
What can the grossing person signal with the ink?
1/ Margins to the pathologist.
2/ Specific areas of interest to the pathologist.
3/ The mode of embedding.
Margins are a controversial issue in a small skin biopsy. Some dermatologists are resentful if a pathologist mentions margins in a shave biopsy, unless they specifically ask for. Nevertheless, grossing person should provide the pathologist with this option in the report. Ink obviously makes it possible.
The main goal of inking in biopsies is to instruct the embedding person about the mode of embedding. If the specimen were cut, it is appropriate to ink because the section area remains unstained. This area should be put down in the mold during embedding. If the specimen were not cut, the inked area should be on the periphery. Otherwise, the most valuable epithelial material can be lost or invisible on the slide.
Ink can point to specific areas of the specimen’s special interest for the pathologist.
Details of inking technique
There are some technical details that make inking efficient or … confusing. They can be summarized as a/ choice of ink, b/ application, and c/fixation of the ink.
Davidson’s Marking System dyes are the commonly used in laboratory practice. Although the color of ink is often a personal preference, there are some considerations in choice of a dye. For example, green almost never interfere with slide stain colors and always visible during embedding. Black ink (India ink in art shops) is protein based that is an advantage because it can be more secure “fixed” to the tissue by cross-linked fixatives; this is also disadvantage for fast processing because the dye is prone to wash off if there was no time for exposure to the dye. Although multicolor inking is rarely applied in small biopsies the rest of dyes can be arranged in the order of less wash off: orange, red (merbromin), yellow(except adipose tissue, but not in lipoma due to the capsule), blue (common laundry bluing).
There is a variety of dye applicators from cotton tipped (rarely used in biopsies) to wooden picks. In biopsies, precise dye application is a priority. It seems, in my experience that flat angled .17 mm smooth, nonwettable Stratagene’s Strata Tips are the most applicable for inking shave and punch biopsies although other similar applicators can be used.
A common practice is to use a “fixer”, or color enhancer, which is incorrectly called mordant (histology definition of mordant is a lake of stain between reagents). Most mordants contain acetic acid, for example, in concentrations of 3% or 50% of white vinegar. In Bouin’s solution acetic acid is a component. 10% acetic acid is the most common practice now. There is unjustified aversion for Bouin’s solution in many laboratories. Some institutions use acetone but it is undesirable for many reasons, including safety considerations.
The ‘fixer” can be applied in different ways, such as using a pipette or sponges (foam polyester urethane to non-latex make-up sponges) after the dyes have been applied. Many laboratories use a squirt bottle, especially for small specimens. This practice seems not optimal due to waste of reagent which is also chemically active irritating the upper airways.
The dyes are more effective when applied to unfixed tissue. Fresh tissue should be patted dry. A common pattern includes the following: dry the tissue vigorously until the paper is not bloated (underestimated step in practice), apply the dye, air-dry for a while, apply a fixer carefully, and dry gently with a sponge, then process.
To cut or not to cut?
Do not cut if
the section will be less than 0.2 cm (difficult to embed straight);
the area of diagnostic interest is too small (less than 1mm) because it might disappear after section (the cut takes tissue and microtomy trimming also);
unfixed specimen less than 0.4 cm, especially with soft/adipose tissue.
Three main conditions are required for taking correct biopsy sections:
1/ Uniform sections –desirable; but impossible without special devices;
2/ Representative – possible; requires ability to present the most diagnostically valuable section;
3/ Informative for embedding- obligatory for the ultimate goal to provide the pathologist with representative pathology material.
A uniform thickness of the section is an obvious requirement for processing but it is difficult to achieve it under conditions of uncertain immobilization of the specimen. The microwave assisted accelerated processing put more demands on uniformity of section. Special device can make uniform 1.5 mm section for X-press microwave assisted processing . The CutMate forceps introduced by Milestone is step ahead in attempts to get uniform preset thickness sections, however, it is completely useless for biopsy section.
A representative section is possible if the grossing person understands diagnostic goal/s of the specimen though not always possible due to variety of the appearance of pathology and some technical conditions of manual section. Sometimes uniformity of section should be sacrificed to avoid unrepresentative section. For example, in a squamous cell carcinoma vs. seborrheic keratosis the section of often fragile lesions should be presented in full that is unachievable without making thicker sections.
An informative unambiguous section prevents the embedding person from guessing how to put the section in the mold without sacrificing the informative material. For example, if the section has both, the area of diagnostic interest and adjusted tissue without such material, the embedding can be wrong by concealing from the pathologist informative material. Figure 1 illustrates a wrong section when the pigmented area is only from one side of the section.
Figure 1. Ambiguous section (the pigmented area is only from one side)
Ink and knife/blade do their part in presenting the most informative diagnostic sample. The ultimate goal of grossing is correct diagnostically sound specimens/sections orientation.
There are different techniques that can be used for proper orientation. Some of these techniques belong to history, some remain in practice. Periodically some forgotten techniques are brought back with corrections.
Many techniques have been employed foe embedding orientation of biopsies from Gelgoam to HistoGel. Two foam polyester pads used to sandwich the specimen in a position for embedding. This method is very popular, however this often does not help the embedding person to correctly orientate a biopsy, especially if it is too small or of uncertain shape. Sometimes the specimen sticks to the upper pad or pops out when the cassette is opened.
A rarely used method of superficial incisions in spongy pads was proposed for small biopsies. Complete through the pad incisions can make this method more universal, especially for skin orientation. The figures 2-4 present orientation embedding for tips in skin excision biopsy.
Figure 2-4. Skin section orientation in the foam pad incision
Correct embedding orientation is the last line where grossing technique can directly influence the specimen processing. The rest is in hands of histotechnologists although wrong grossing technique like poor fixation, wrong thickness of the section, and incorrect placement in the processing cassette could compromise cutting and staining process.
Every sampling is accompanied by gross description (See in the end of Introduction to Skin Grossing chapter).
The general principles and techniques will be repeated in special chapters (shave biopsy, punch biopsy, and excision). There are specifics in details of grossing in every type of skin biopsy that should be addressed.