|Year : 2018 | Volume
| Issue : 2 | Page : 63-67
Grossing of tissue specimens in oral pathology - Elemental guidelines
Ruthushree Theresa, M Harsha, Vikram S Amberkar
Department of Oral and Maxillofacial Pathology, Rajiv Gandhi University of Health Sciences, College of Dental Sciences, Davangere, Karnataka, India
|Date of Web Publication||18-Dec-2018|
c/o Dr. Rohit Barnabas, Block 62 House 2D2, Christian Medical College Vellore - Nursing College Campus, Kagithapatrai, Vellore - 632 012, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Grossing is a gross examination of surgical specimens in which pathologists inspect the excised specimen with the bare eye to obtain diagnostic information. It refers to the examination and dissection of surgical specimens, along with preparation of sections from those tissues requiring processing, and is the initial step in surgical pathology dissection. Careful handling of the tissue with proper fixation and tissue processing will enable a confident histopathological diagnosis.
Keywords: Fixation, grossing, processing
|How to cite this article:|
Theresa R, Harsha M, Amberkar VS. Grossing of tissue specimens in oral pathology - Elemental guidelines. Int J Oral Health Sci 2018;8:63-7
| Introduction|| |
Gross examination of pathological tissue specimens forms an important part in reaching at a correct diagnosis. Accurate gross description and observation of the pathology specimen can give many clues to aid in the final diagnosis. The final report must include macroscopic and microscopic findings along with the final diagnosis.
Grossing is a gross examination of surgical specimens in which pathologists inspect the excised specimen with the bare eye to obtain diagnostic information.
Grossing refers to the examination and dissection of surgical specimens, along with preparation of sections from those tissues requiring processing, and is the initial step in surgical pathology dissection. It is an initial and most important step in surgical pathology for obtaining accurate diagnosis.
The gross room is the area where pathology specimens from the operating rooms (ORs) are transferred for pathological review and analysis. Thus, it serves as the bridge between the surgeon and diagnostic surgical pathologist in that a correct diagnosis or treatment of a patient depends on the proper handling and processing of the specimens of tissue transferred to this area.
The room should be large, well illuminated. It should be properly ventilated with an exhaust fan. It should contain shelves for specimen containers, ready access to formalin, large table for dissection of specimens, and sink with provision for water. Other facilities ideally required are photographic facility, X-ray unit with view box, and refrigerator.
The biopsy grossing triage facilitates optimal handling of processing starting at specimen receipt. Many mistakes, besides specimen misidentification, occur at the initial preanalytical phase during specimen accession. The grossing person is instrumental in managing the accession process. Although very often neither pathologists nor pathology residents are closely involved in biopsy triage, it is important to understand the process for effective control of this phase of grossing.
Besides “separation of workload” when a laboratory directs different case types, there are three main points for considerations during biopsy grossing triage the specimen's category (i.e., whether it is a cytology or a surgical pathology sample), determination of the priority for processing, and securing appropriate specimen preservation for processing. Effective triaging of the specimen requires knowledge, experience, and common sense. Labeling in the OR is critical for successful and timely processing of biopsy material.
| Procedures Followed during Grossing|| |
Labeling the specimen
Specimen labeling is an important step before proceeding with the grossing procedures. This aids in facilitates in proper identification of the specimen. Most institutions have their own unique way of specimen identification giving each patient and tissue unique accession numbers which usually include the year the specimen was collected with varying prefixes for different types of specimens.
The most important step in specimen handling is the correct identification of the specimen(s) with unique numbers so that an accurate link between the specimen and the patient from whom the specimen was removed is maintained. Each specimen container should include the patient's name with age (birth date) and a medical record number along with matching paperwork (e.g., a surgical request form). These labels must be consistent and should be on the container so that the labels cannot be separated from the specimen (e.g., labels should not be attached just to the top of the container). The surgical request form provides the actual request for pathological services and the required relevant clinical history of the patient. Any discrepancies in specimen identification/labeling (e.g., a medical record number that does not match the patient name or a surgical request form which does not match the specimen) must be resolved with the clinician/surgeon before processing any specimen, and the discrepancy should be noted on the request form. Misidentification of any specimen can result in failure to make a proper diagnosis on one or more patients, incorrect treatment, and possibly legal action.,
Fixation of the specimen
After receiving the specimen from the surgeon, the specimens have to be fixed in an ideal fixative for preventing it from autolysis or damaging of the specimens. Routinely, 10% neutral-buffered formalin is the most common fixative used for tissues submitted for examination including tooth specimen. However, mineralized samples such as bone or tooth may require decalcification before it can be processed. To facilitate uniform penetration of fixative, it is necessary to fix small volumes of tissues (5 mm–1 cm). The volume of the fixative should be more than 20 times the volume of the tissue. In practice, it is assumed that these processes require at least 1 h per mm of tissue thickness, but routinely, the tissues are fixed for 24–48 h. It may be necessary to replace the fixative with a fresh solution when the specimen contains a high percentage of blood. Covering large specimens with fixative-soaked gauze or cloth may help penetration and reduce surface drying.,,
| General Requirements for Biopsy Grossing|| |
Completeness of the sample
Complete, representative, and informative sampling
Complete (or entire) – all the material must be submitted entirely using all means of grossing available. Representative (or adequate) – the submission should present the most diagnostically valuable parts of the biopsy specimen (unfortunately, entire submission does not guarantee that the most diagnostically important area will appear on the slide).
Every biopsy grossing description in the pathology report must include the statement “entirely submitted.” This is the fundamental rule of grossing small biopsies. The completeness of submission depends to great extent on the grossing person, especially if the specimen contains numerous fragments or uncountable material. There is a substantial difference between the completeness of submission and completeness of presentation of the material under the pathologists' microscope. If technological details are not taken into account, the “entirely submitted” statement may become an empty phrase. The grossing person should have an understanding of the process of embedding and create the conditions for complete submission of the specimen.,
| Specimen Photography and Radiography|| |
Photographs of fresh or fixed anatomic pathology specimens are obtained to aid in the documenting of pathologic lesions. To meet the needs of the medical staff for conferences, teaching, and patient care activities, photographs of gross surgical and autopsy case specimens are taken. Color pictures are best taken either fresh or after brief (few to 10 min) fixation. The brief immersion reduces distracting glare from reflected light. The color though may be altered if the immersion into formalin is prolonged (hours or days). If immersion is prolonged, the return of some color can be achieved by placement in 70% ethanol for 10–15 min. Before photographing, the tissue must be prepared by washing to remove blood, blood clots and fat, opening the ducts and vessels, and removing other unnecessary tissues around the lesion. The background should be spotlessly clean with no texture and should be well illuminated. The use of drapes, sponges, and gauzes should be avoided. Distracters such as hands, forceps, probes, scissors, and paper clips should be removed. Reflective glare should be avoided by properly placing the illumination system, by turning off the room lights, and by blotting the cut section of the specimen with gauze. Several photographs of the lesion using different exposures are recommended. A clean ruler with metric system should be used for obtaining a reference to size. The specimen should be properly oriented in the anatomical position and centered.,
Photographs of the external surface of the intact specimen as well as the cut surface should be taken. If possible, include normal structures in the photograph to serve as a frame of reference for the lesion.
Radiographic examination of surgical specimens sometimes provides important information. Specimens suitable for this type of examination include bone lesions, calcified soft-tissue masses, and lesions with embedded tooth radiopaque foreign bodies such as metal clips. Some pathologists have found specimen X-ray films useful for locating lymph nodes in radical resection specimens. Others have used them to perform a microradiographic analysis of the bone.
| Sectioning of Specimen|| |
Any cut parallel to the longest dimension of a structure produces a longitudinal section, and any cut that is perpendicular to it produces a cross section. A cut at any angle between these two planes produces an oblique section. A cut through the middle of a spherical structure produces a cross section and one that only grazes the surface produces grazing section, otherwise known as tangential section.
In some cases, the internal microscopic structure of parts of the body is so complicated that it can only be understood by mounting photographic enlargements of serial (consecutive) sections on the material of appropriate thickness and assembling them in a proper order to constitute large reconstruction.
| Inking|| |
The most popular method to make visible small fragments of tissue to the embedding person is to mark them with hematoxylin or a combination of eosin Y and phloxine B (10:1) or Indian ink. For extremely small fragments, besides marking a warning on the cassette, a procedure that can be called dye crystallization may be helpful. By applying layers of hematoxylin, the minuscule fragment is “incorporated” in the dye that is taken away by the initial trim by the microtome blade. This method has been tested in many specimens, including minuscule vocal cord or brain biopsies.
Separation of the material into different cassettes can also be very helpful if the size of the fragments is dramatically different, especially when a very small fragment accompanies other bigger visible fragments. The simplest and most commonly available method is wrapping the specimen in a cassette-sized lens paper. Some details count. The paper should be wet, wrapped in an envelope like squares with the specimen directly in the center.
There is always the possibility of contamination of the specimen with extraneous materials (the so-called floaters) independently of the kind of transfer device used. Floaters are never welcome by pathologists not only because they are a nuisance but also they represent a liability. Although the main culprit for floaters is the embedding area (contaminated wells in the embedding stations, forceps in paraffin with stacked miniscule fragments, etc.), grossing can also contribute to specimen contamination with extraneous tissue (“cutting board metastasis”) the most important grossing skills are the ability to choose the most diagnostically valuable parts of the tissue for the pathologist to view on the microscopic slide and the ability to provide informative, unambiguous instruction to the embedding, and microtomy personnel for the orientation of the biopsies. Both of these components of grossing are inseparable.,
If the ink does not carry any message, it does not make any sense to ink. Ink can obscure the pathologist's vision when reviewing the histologic sections. In thin specimens, 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?
- Margins to the pathologist
- Specific areas of interest to the pathologist
- The mode of embedding.,,
| Processing of the Specimens|| |
The processing of tissue includes dehydration, clearing, and embedding steps. Dehydration involves the removal of fixative and water from the tissue and their replacement with dehydrating agents by placement in increasing concentrations of ethanol. Next, the clearing step involves replacing the dehydrating fluid with a lipid solvent (e.g., xylene). The tissue is subsequently removed from the cassette and placed in a molten wax-filled mold for embedding. At this point, the orientation of the tissue within the mold is critical, as it will determine the plane through which the section will be cut. Incorrect placement of tissues may result in diagnostically important areas being missed or damaged later.
| Orientation|| |
Tissue should be oriented to determine the depth of invasion of the lesion and the margins of resection. The ultimate goal of grossing is correct, diagnostically sound sections with proper orientation. Various techniques have been used for orientation such as sticking specimen to gel foam using cyanoacrylate or strips of cellulose acetate. Agar, dehydrated plane cucumber slabs, and histogel can also be used. In case of incisional biopsies, the specimen may be orientated by placing a suture at a known margin, for example, the anterior or superior margin. This would enable the pathologist to confidently indicate the precise location of any residual tumor. The same applies to surgical resection specimens.,
| Embedding Sectioning and Staining|| |
Correct embedding and orientation is the last step where grossing can directly influence specimen processing. Silicone pads for automatic embedding orientation are also proposed, but they are still at the stage of testing. The rest is in the hands of the histotechnologists who do the cutting and staining, although poor fixation, wrong thickness of the section, and incorrect placement in the processing cassette can significantly compromise the cutting and staining process.
The waxed block with the specimen is next placed in an instrument with fine blades called a microtome. Rotation of the drive wheel moves the block holder a controlled distance forward, the blade's edge strikes the tissue block, and thin sections are cut and affixed to a glass slide. Sections are usually 4–6 μ thick so that a single layer of cells can later be seen under the microscope. The following thorough drying of the tissue sections is ready for staining.
As most tissues are colorless, methods of staining tissues have been developed to make them visible while also allowing distinctions to be made between tissue components. This is done using mixtures of acidic or basic dyes that selectively stain various tissue elements. The constituents that react with basic dyes do so because of acid in their composition (e.g. nucleoproteins) while acidic dyes stain basic tissue components (e.g. cytoplasmic proteins). Of all routine stains, the combination of hematoxylin and eosin stain is the most commonly used stain. This stain is considered the gold standard in histology, and in a typical tissue section, nuclei are stained blue/purple, whereas the cytoplasm and surrounding matrix have varying degrees of pink staining. Therefore, the hematoxylin and eosin stain has the ability to reveal structural information with specific functional implications. Special stains use a slightly different technique to stain particular structures.,
When routine or special staining cannot provide all the diagnostic answers required, histopathologists can use advanced staining techniques including immunohistochemistry or in situ hybridization. Finally, the stained slides should be mounted with an appropriate mounting media and coverslip. The slides can be viewed under microscope for providing diagnosis.
| Health Hazards and Safety Measures|| |
Staff members working in the gross room area encounter many possible risks including infections, chemicals which may be flammable, toxic, allergenic, or carcinogenic, electrical and physical hazards as well as cuts and needle stick injuries; the most common hazard being needlestick injuries. Bone dust as well as bone fragments and crumbles disseminated in the working environment are potentially biohazardous and carcinogenic. Formalin fumes are also known to be a health hazard. It is a severe eye and skin irritant and is toxic by ingestion and inhalation. These can be minimized by proper tissue handling and fixation of the specimen before grossing. All tissues must be considered potentially hazardous, and universal precautions must be taken as per occupational safety and health administration regulations. Adequate protective measures to protect from infection must be undertaken such as disposable gowns, gloves, facemasks, and eye gear. Contact with chemicals should be minimized, and the protective gear should be dispose off in the correct manner. The laboratory personnel should clean the instruments and wash hands regularly to avoid spread of infection.,,
| Conclusion|| |
There are seven major components in processing a gross specimen:
- Reliable and rapid transfer of the specimen from surgery to pathology
- Accurate identification of all specimens
- Accurate description of original specimens
- Accurate description of additional specimens received from the same patient – operation
- Recording normal and abnormal features of the specimen including markers (e.g., sutures) which orientate the specimens
- Special studies requested and/or needed
- The location from which specific sections of tissue are taken for histologic evaluation.
If and only if specimens are properly grossed, a surgical pathologist expects proper processing. In today's busy medical care system, it is not only the pathologist but also residents, pathologist's assistants, and trained histotechnologists who may be grossing specimens, all work together to achieve a common goal of optimal patient care. Careful handling of the tissue with proper fixation and tissue processing will enable a confident histopathological diagnosis.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Geller SA, Horowitz RE. Gross examination. Methods Mol Biol 2014;1180:3-19.
Peters SR, editor. A practical guide to frozen section technique. Springer Science & Business Media; 2010. p. 13-15.
Shashidara R, Patil S, Rao RS, Scindiya E, Tryphena A. Grossing of oral pathologies-revisited. J Contemp Dent Pract 2017;18:1213-22.
Rao RS, Premalatha BR. Grossing in oral pathology: General principles and guidelines. World J Dent 2010;1:35-41.
Dimenstein IB. Grossing biopsies: An introduction to general principles and techniques. Ann Diagn Pathol 2009;13:106-13.
Bell WC, Young ES, Billings PE, Grizzle WE. The efficient operation of the surgical pathology gross room. Biotech Histochem 2008;83:71-82.
Vaught JB, Henderson MK. Henderson: Biological sample collection, processing, storage and information management. IARC Sci Publ 2011;163:23-42.
Hassan U, Baig MK, Mushtaq S. importance of pH of fixatives used for fixation of histopathology specimens – An un-recognized issue. J Islamabad Med Dent Coll (JIMDC) 2015;4:103-5.
Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 2002;161:1961-71.
Hruban RH, Westra WH, Phepls TH, Isacson C. Surgical pathology dissection. An illustrated guide book. 2nd
ed. Springer; 2003. p. 2-15.
Rampy BA, Glassy EF. Pathology gross photography: The beginning of digital pathology. Surg Pathol Clin 2015;8:195-211.
Khurana JS, Vivian Arguello-Guerra. Grossing of bone soft tissue (common specimens and procedures) 2nd
ed. Springer: Humana press; 2009. p. 125-8.
Breast PA. In: Westra WH, Hruban RH, Phelps TH, Isacson C, editors. Surgical Pathology Dissection. An Illustrated Guide. 2nd
ed. New York: Springer-Verlag; 2003. p. 132-41.
Parkinson AV, Cannon CR, Hayne ST. Color coding surgical margins with the Davidson marking system. J Histotechnol 1990;13:293.
Pursnani D, Arora S, Palur K, Ambica C, Yelikar BR. Inking in Surgical Pathology: Does the Method Matter? A Procedural Analysis of a Spectrum of Colours. Turkish Journal of Pathology 2016; 32:112-8.
Ham WA, Cormack DH. Ham's Histology. 9th
ed. Philadelphia, PA: JB Lippincott Company; 1987. p. 3-23.
Bancroft JD, Gamble M, editors. Theory and practice of histological techniques. Elsevier Health Sciences; 2008. p. 95-100.
Swan RW, Davis HJ. The biopsy-cucumber unit. A method to improve tissue orientation. Obstet Gynecol 1970;36:803-5.
Riddell R, Jain D. Lewin, Weinstein and Riddell's Gastrointestinal Pathology and its Clinical Implications. Lippincott Williams & Wilkins; 2014. p
Diederichsen C, Whitlatch S. Description and preliminary results of a novel cassette system (Tissue-tek Paraform Cassette System). HistoLogic 1999;31:28-30.
Thirumala S, Pinkhasov DP, Medalie N. A simple yet effective technique to improve laboratory safety for the grossing of large surgical specimens. Ann Diagn Pathol 2000;4:44-5.
Dimenstein IB. Bone grossing techniques: Helpful hints and procedures. Ann Diagn Pathol 2008;12:191-8.