Selected biopsy grossing techniques cover some areas which are inadequately represented in the surgical pathology grossing literature, including the current main source as the Lester’s manual. All materials reflect my personal experience therefore they have some bias. Although different practices might apply slightly different approaches, these materials try justifying the suggested methodology.
General principles of biopsy grossing techniques are presented in the article Dimenstein IB: Grossing biopsies: an introduction to general principles and techniques. Annals of Diagnostic Pathology2009; 13: 106-113 and in this website’s the Grossing in Dermatopathology manual. However, a standalone description of common principles would be useful for selected biopsy grossing techniques.
Definition of biopsy: the removal for histology examination of tissue structure from living body for diagnostic purposes.
Biopsy grossing starts at specimen receipt. Triage facilitates optimal handling during specimen accession. Besides workload considerations in separation of specimens, there are three main points of grossing triage:
Category (surgical pathology, cytology, microbiology, molecular biology etc.);
Priority (rush, processing workflow);
Preservation (fixation for regular processing, reagents for ancillary studies).
The modern biopsies processing includes many ancillary studies which require different modes of reservation. Fixation is only one though the most ubiquitous technique of preservation.
The table 1 summarizes most frequent biopsy preservations methods although this table is incomplete due to the variety of studies and reagents.
able 1 Biopsy preservation methods
|Type of preservation||Reagent/s||Purpose||Special conditions|
|Transport isotonic liquid||Normal saline (0.85% NaCl)||Prevent from drying||Short term of exposure due to maceration and autolysis|
|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)|
|Nutrition medium||RPMI 1640 (Roswell Park Memorial Institute) medium||Flow cytometry for immunophnotiping||Preferable sterility to preserve antigen integrity|
|Nutrition medium||DMEM-F-12 (Dulbecco’s modified Eagles’s medium) contains antibiotics and 10% fetal bovine serum||cytogenetics||Maximal sterility for cell culture|
|Freezing||Liquid nitrogen, isopentan||Molecular biology, muscle, nerve biopsies||Snap freeze|
|Transport medium||ThinPrep Test||Cell preservative solution||Prompt use|
As a variant of preservation, fixation has a dual role. Besides its main purpose to preserve cellular structures, it serves so called hardening of the specimen that is important for grossing manipulation. Hardening is an old term that stems from pre-formalin era of alcohol fixation. Actually fixation provides immobilization of the specimen’s tissues by preventing them from moving during cutting procedure.
In 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 in higher temperature, vacuum and pressure processors, 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. Formation of acid formalin hematin makes blood turn tan- brown. This is important in biopsies which arrive at different stage of fixation. When the red-pink dots of the areas of biopsy removal disappear by turning tan, the specimen can be considered ready for tissue processor although the fixation is continued in the dehydrating alcohols. Many biopsies, for example needle biopsies, do not have these clues which require additional time for fixation just in case. The current requirement for Her-2 assays of not less than 6 hours is another example of importance to adhere to terms of fixation.
TECHNIQUES in GROSSING BIOPSIES
Sampling, or cut-in, is the main part of 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.
General requirements to biopsy grossing technique: complete, representative, informative.
Complete, or entire – all material must be submitted entirely using all means of grossing;
Representative, or adequate – the submission 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 further processing during embedding and microtomy.
The fundamental rule of grossing biopsies is in completeness of the submission. It depends solely on the grossing person, especially if the specimen contains numerous fragments or uncountable material. Every biopsy grossing description in the pathology report must include a statement: “submitted entirely.” If technological details are not taken into account “submitted entirely” statement is an empty phrase.
The most popular method to make visible small fragments is to mark them with Hematoxyline or combination of eosin Y and Phloxin B (10:1).
In extremely small fragment, besides marks of warning on the cassette, a procedure that can be called dye crystallization is helpful. By putting layers of Hematoxyline, the minuscule fragment is “incorporated” in dye that is taken away by initial trim by the microtome blade. This method has been tested in many specimens, including on vocal cord or brain miniscule biopsies.
Separation in different cassettes is useful, if size of fragment is dramatically different, especially when a very small fragment accompanies big visible fragments. This often occurs in cervical biopsies.
Completeness of biopsy representation is especially important in breast needle biopsy owing the heterogeneity of the material. Number of cores is substantial because the pathologist should be confident that all cores are examined. Cores should be aligned in parallel on a flat surface during grossing and repeated in the embedding mold. Prostate needle biopsies are another example of heterogeneous core material that requires special techniques to provide completeness of submission for processing.
All generations of biopsy grossing practitioners tried to use different means to provide completeness of biopsy submission. These attempts can be approximately divided in two categories: “gadgets” and techniques (Table 2).
Table 2. Methods for complete biopsy submission
|Lens paper wrapping||Filtration|
|Nylon 0.2 mm mesh, paper bags||Cell blocks|
|Foam pads||HistoGel, agar-agar|
|Special cassettes||Dye crystallization|
The simplest and most available is wrapping the specimen in a cassette size lens paper, but is necessary to mention some details. The paper should be wet, wrapped in envelop like squares with the specimen directly in the center, better circled with a red pencil (Chinese pencil, for instance) especially if the specimen is small. Unwrapping poorly bend paper in not only a pain in the neck for embedding but also valuable material might be lost in folds.
Some laboratories use Obex round papers folded as cones. Tea bags (Salada tea) can be used because they are made of long-staple abaca fibers. Some histotechnologists use Jumbo End Wraps from beauty store (very cheap!), especially for cell blocks. The popular and convenient nylon cloth 0.2 mm mesh diameter bags, as well as porous paper bags, have unfortunate deficiency that the specimen or part of it pops out that can cause loosing valuable material.
Nylon Tissue Biopsy Bags ar offered by Merrick Inc. (Fig.1)
There are numerous fenestrated mesh processing cassettes which have filtration abilities. They look attractive and in many instances convenient but the problem is how to get the completely submitted specimen in the embedding mold. In opposite to the lens paper and nylon bags when with one movement of a blade the content in the melted paraffin goes in the embedding mold completely, the specimen ought to be transferred from the edges of the fenestrated cassette that is technically difficult. By the way, biopsy cassettes can trap air causing they float.
Figure 2. Fenestrated cassette .
The fenestrated cassettes are continuing in development of the design. Very interesting option provides Vortex ™ Cassettes. More oval and smooth inside makes easier to embed with assurance of submission’s completeness.
Ubiquitous cellular polyester urethane blue foam pads (can be different colors) are the most popular tools for providing completeness of a biopsy submission in the cassette when small samples are sandwiched between two pads and placed in a cassette or in tissue capsules. Simple and convenient for embedding, they have some deficiencies as far the completeness is concerned. For example, they are used in prostate needle biopsies, however, the cores in vast majority of cases are different in size and thickness. Some of them can be trapped in the holes of the foam under condition of vacuum and pressure tissue processors. There are observations of artifacts, especially if the biopsy has not been fixed enough. Artifacts, as well as loss of the material occur if the pad is dry or the biopsy has mucinous content. The macro waive vacuum assisted processor’s manuals also object their use for carried over consideration that is a short term processing should be taken in consideration. Again, as little as possible, but they are indispensable in dermatopathology orientation. Hopefully, they will be replaced by other methods in the future.
There are no questions lens paper vs. sponges or fenestrate cassette vs. nylon bags and so on. Everything depends on the concrete grossing situation and specifics of the specimen. However, the foam sponge should be avoided as much as possible. Actually, I would leave them for orientation purposes only. Suggestions for Telfa™ pads looks unreasonable technologically because they are not tailored for a processing cassettes, besides that they are impregnated by an untested antimicrobial chemical component.
While working with bags, pads, and special cassettes, in this author’s experience, filtration directly in the processing cassette is the best method if there are either numerous fragments or scarce, especially mucous, materials. The importance of filtration is underestimated in grossing biopsies.
A wet baby diaper pad, as an absorbing material, under the cassette and moistened porous lens paper like fabric inside the cassette can provide effective filtration. This fast method assures complete submission of the biopsy with minimal use of instruments, preventing any contamination. See “The Diaper Pad for Filtration in Surgical Pathology” at the link “Equipment, Instruments, Gadgets”.
Cell block as a routine in cytology, often is used in biopsy grossing if there is scarce material that can be lost in processing if the standard processing is applied. The most often this method is appropriate if the biopsy is taken with a brush (endobronchial, urethral). Although fixation is an impediment for cell block method, intensive spin can provide diagnostic material.
HistoGel tissue processing medium can be used for complete submission extremely minute specimens, for example brain biopsies. This method now replaced the old method of keeping small biopsy fragment in agar-agar . I do not have an experience with this method but met many histotechnologists who work in neuroscience with positive referrals.
Dr. Ervin Shaw http://www.palpath.com/prosca.htm used a color coded pre- embedded agar technique to help arrange cores of prostate needle biopsies specimens for orientation flat during grossing in 90th. This allows them to process three cores in one block and still separately identify each core and to orient them from more peripheral to central. This additional information may be helpful to some clinicians, including radiologists using endorectal-coil MRI. Dr. Shaw used the agar technique to help keep small biopsies on edge for the entire processing and paraffin embedding (personal communication).
Cucumber method attached small biopsies to squares of cucumber, which had been preliminary dehydrated in several changes of alcohol with a egg-albumin/glycerol mixture. The biopsy/cucumber unit was embedded together after processing and cut and stained as one unit. This method was used rarely for cervical biopsies (very small), but more frequently for gastric and especially duodenal biopsies. It looks that now it was abandoned. In general, the method is good, though cumbersome, but in practice, often cucumber and the biopsy arrived in one container but separate that made the idea of orientation by the clinician a mockery, although this could be fixed by a grossing person who understand the process. In principle, this method can be used, although there are other organizational issues. I doubt that this method can be used now in the assembly line biopsy production.
Transfer instruments and devices Although in the vast majority of biopsy grossing situation the forceps, or different kind of them depending on the size of the specimens and conditions, are used in transferring biopsy into processing cassette, there are situations when other instruments or devices can be employed. The ideal would be direct filtration from the container into cassette that most appropriate in endometrial, cervical curettage, or bone marrow aspirate, but even in this situation sometimes forceps is used to assure completeness of submission.
In a case of numerous fragments, as in gastrointestinal biopsies, a disposable transfer pipette, like S/P Plastic Transfer Pipet, is cheap, fast and reliable. In rare occasions when the film like specimen is difficult to handle, an inoculating loop or nichrome wire loop like device can be used for reliable transfer of the specimen from the container into cassette (of course, using the lens paper to wrap up).
Completeness of submission depends also on clinicians who send the material. The common habit, for example, to send curettage samples on napkins is wrong. Clear product should go to pathology. Some napkins deteriorate in normal saline, as well as in formalin. The specimen slides to the fringes, steaks there. Under these circumstances, it is difficult to recognize the specimen and to take it out. Endometrial curettage as well as vocal cord polyps in SafeTouchTissueTrap cannot be accepted as biopsy. It is difficult, almost impossible to get out all material from the holes of the container. Now colonoscopy practice uses often Suction Polyp Trap to collect polyps in capture chambers with perforated bottom, and some of the material sticks in the perforation so tight that there is no way to take them out without damaging the tissue. And, of course, already mentioned mode of sending material in dry condition is the path to lose biopsy material.
Extraneous tissues, or floaters
The completeness of submission has its opposite as extraneous tissues, “floaters.” In biopsies floaters might be not only a nuance but liability under condition of modern surgical pathology with numerous very much similar biopsies. Grossing has it significant share as culprit of this unfortunate error of processing. A set of preventive measures (cleaning and changing the processing board, instruments, and gloves) must be implemented. Floaters problem requires a comprehensive approach starting from specimen receipt to histology stainers, but grossing is one of the main areas of contamination that might have significant diagnostic consequences. Details of preventive measures are presented somewhere else. See “ Extraneous Tissues in Surgical Pathology Practice” at the link “Perspectives in Surgical Pathology”.
REPRESENTATIVE and INFORMATIVE
Both of these components of grossing are inseparable. Ink and knife are means for nonverbal communication between the grossing person and the pathologist, as well as the embedding person and histotechnologists.
If the ink does not carry any message it does not make any sense to ink. Ink can obscure the pathologist’s vision. In thin specimen insoluble ink fragments can diffuse from the area of inking. 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 questionable issue in a small 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. For example, an inked dot at the subepithelial area in a cervical biopsy gives the embedding person definite instruction that during embedding the dot should be at the periphery (Figure 3). 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. For example, area of merging of ectocervix and endocervix in cone biopsy (Figure 1).
Figure 3. Two color inked cervical cone biopsy
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 were 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).
I have read about tattoo ink as a marking tool, but haven’t seen any facility, which used them. I do not think that they will be cheaper than traditional vendors charge if you by small quantities. However, the main thing is that we do not need an intrinsic involvement with the inked tissue. Surgical pathology uses margin and orientation marking inks. Tattoo ink is intended to stay forever, for some users unfortunately.
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 that cotton tipped stick are not appropriate completely in biopsies because they are not precise and have threads which can be seen under the microscope give additional difficulties to the pathologist. In the author’s experience, that flat angled .17 mm smooth nonwettable Stratagene’s Strata Tips are the most applicable for inking biopsies although other similar applicators can be used (Figure 4).
Figure 4. Plastic ink applicators
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. 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.
The dyes are more effective when applied to unfixed tissue. Fresh tissue should be patted dry. The common pattern includes: dry 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 understanding pathology issues and 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. Or adenomatous villous colon polyp requires special technique of sections to preserve integrity of the specimen by maintaining the relationship with the stalk in most informative way. Or a cervical cone biopsy requires certain cutting technique to present to the pathologist sections that reflect ecto/endo cervical transitional areas.
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 5 illustrates a wrong section when the pigmented area is only from one side of the section.
Figure 6. 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. One technique involves sticking a specimen to slabs of Gelfoam with cyanoacrylate. A different technique arranges a duodenal biopsy mucosa upwards on small strips of ruled cellulose acetate. Agar-agar has been used for orientation of biopsies and small specimens. Dehydrated slotted or plain cucumber slabs have been initially recommended to mount cervical but now remain predominately for gastrointestinal , especially duodenum , sometimes for laryngeal biopsies .
HistoGel tissue-processing medium can be used for specimen orientation, for example brain needle biopsies.
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 orientation skin, cervical, and gastrointestinal polyps. The figure 7 presents orientation of a biopsy.
Figure 7. Biopsy orientation in the foam pad incision
The search for methods of specimen’s orientation continues. There are proposals of cassettes with fluoropolymer section able immobilizing porous platforms or silicone pads for embedding orientation, but they are still at the stage of testing.
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.
The general principles and techniques will be repeated in surgical pathology subspecialties (gastrointestinal, gynecology, etc.). Dermatopathology is presented in a separate manual “Grossing in Dermatopathology.” There are specifics in details of grossing in every subspecialty that should be addressed. Selected biopsy techniques will be presented in separate postings. They reflect personal experience.