Specimen Processing: From Gross Specimens to Tissue Cassettes

Specimen Identification

Pathology departments should assign each case a unique identification number that includes the year (e.g., S-22-4382 for a specimen received in 2022). This number is used to identify all specimen containers, additional materials (e.g., specimen radiographs, slides, and reports from intraoperative consultation), and the requisition form. Each “case” is usually defined as all specimens derived from the same surgical procedure. For example, five skin biopsies from the same patient, performed on the same day, would be given the same pathology number.

The first step in specimen processing is identification of all the components. The specimen container label must include the patient’s name and date of birth or the patient’s assigned hospital or clinic patient identification number. The name or number is matched with any accompanying paperwork. The number and types of specimens received are checked against the list provided on the requisition form. Additional parts of the specimen generated by the pathology department (e.g., frozen section remnants or tissue taken for special studies) are also identified.

Any inconsistencies in labeling or missing specimens must be resolved the same day when memories are fresh and when it may be possible to recover a misplaced specimen or acquire a new specimen. The clinician submitting the specimen should be contacted as soon as a problem is found. If the clinician cannot be reached, the call and the time it was made should be documented. The specimen should be kept intact (but in fixative, if possible) until any issues are resolved.

Patient History

For optimal specimen processing, it is essential for the prosector to know why the specimen was removed from the patient and the goals for examining the specimen. Ideally, sufficient history has been provided by the clinician on the requisition form. For large complex specimens, the number, location, and size of expected lesions and whether or not the patient has been treated prior to surgery is required information for optimal specimen evaluation. The usual reasons for surgery and the most common lesions are discussed for each specimen type in subsequent chapters. If it is unclear why a procedure was performed, it is always preferable to contact the clinician before proceeding.

Gross Examination and Dissection

Each specimen is approached with clear goals in mind based on the type of specimen and the reason for the surgical procedure. If it is a photogenic specimen or photography is recommended (e.g., medicolegal cases), consider the best method to illustrate the pathology before inking or dissecting (see Chapter 7 , Microscopy and Photography).

• Identify all anatomic structures present. This might include determining the parts of the bowel present, the lobe of lung, or muscle, bone, and nerve included in an amputation for tumor. Diagrams in the sections on specific specimens illustrate the anatomic components of large resections.

• Orientation markers. Anatomic (e.g., an axillary tail on a mastectomy) or surgically designated (e.g., a suture) orientation marks must be identified. These landmarks should not be obscured or removed during dissection if they are necessary for orientation. If a landmark must be removed, the site can be identified by colored inks, sutures, or nicks in the attached skin.

At times, radiologic studies, operative notes, or additional information from the surgeon can aid in understanding the orientation. If orientation is unclear (e.g., an unoriented simple mastectomy) from gross examination and the information available, the surgeon should be called to request additional information.

• Measurements. Dimensions (in metric units) and, for some specimens, weights should be recorded on intact specimens prior to dissection and fixation.

• Inking margins. Small biopsies for non-neoplastic disease (e.g., colon biopsies), incisional biopsies of tumors, or large specimens for non-neoplastic disease (e.g., diverticulitis) are not usually inked. Some institutions find that inking small specimens (such as skin or core needle biopsies) is helpful to the histotechnologist when embedding or sectioning these specimens. In addition, the ink color can help identify specimen mix-ups if different colors are used for similar specimens received on the same day as the ink color on the slides can be matched to the color in the gross description.

Small simple specimens with known or potential neoplasias are often best inked in their entirety before proceeding (e.g., primary breast biopsies or the margins of skin excisions for pigmented lesions). All margins with areas of gross tumor involvement in large resections are inked. However, for large complicated resections with grossly negative margins, it may be better to delay inking until the closest area of the tumor to margin is identified after sectioning. Globally inking large complicated specimens may obscure anatomic landmarks and can increase the likelihood of artifactually introducing ink into tissue that is not present at the margin.

• Dissection. No specimen is adequately examined until it has been completely dissected and serially sectioned. Although there are advantages to keeping specimens relatively intact, this is not an excuse for a limited and inadequate examination. With experience, specimens can be thoroughly sectioned without rendering them unrecognizable or incapable of orientation.

The initial examination is simplified by opening all hollow structures (e.g., bowel sections for neoplasia and uteri) except in cases in which inflation provides better preservation (e.g., bladders and colon resections for diverticular disease). For cases with tumors, the examination is directed toward determining the site and size of the tumor, location, and identity of structures invaded by tumor, vascular invasion, distance from resection margins, and the presence of lymph nodes in the specimen. For other specimens, identification of the suspected disease process (e.g., chronic cholecystitis and cholelithiasis), any incidental findings (e.g., serosal tumor implants on a cholecystectomy specimen), and the identification of abnormal lymph nodes are important.

• Identification of pathologic processes. All pathologic lesions have characteristic gross appearances. The chapters on specific specimens provide gross differential diagnoses of common lesions. If a lesion reported to be present, or previously diagnosed by biopsy, cannot be found (e.g., a fistula tract or avascular necrosis of the femoral head) or if the lesion is unusual in appearance, it is advisable to consult with the surgeon before further processing of the specimen. It is important to document the absence of a lesion if the surgical intent was to remove the lesion (e.g., the absence of a biopsy cavity in a breast re-excision specimen or the absence of a large polyp in a bowel resection).

• Histologic sections. Sections are taken that best demonstrate the features seen on gross examination, not simply random sections. For example, the best section demonstrating penetration of the bowel wall by a colon carcinoma is the one showing the deepest extent of tumor. To find this area, the entire carcinoma must be carefully sectioned. Similarly, margins must be taken at the sites most likely to show tumor at the margin.

Residents and Pathology Assistants should request assistance from more senior pathologists for the following types of specimens:

• Complex (e.g., with multiple organs)

• Difficult or ambiguous orientation

• Unusual or unexpected gross appearance

• Difficult to interpret/reconstruct after sectioning

• Indication for surgery and/or purpose of the pathologic examination unclear

This practice will enhance the learning experience, as well as facilitate the optimal evaluation and sign-out of the case.

It is very difficult for a senior pathologist to sign out a case that has been randomly sectioned by a prosector who is unaware of the reason for the pathologic examination. If a picture is worth a thousand words, a good gross examination is better than a thousand slides!

Lymph Nodes

Lymph nodes are the most important component of all tumor resections! Gross primary tumors tend to distract the prosector, as the tumor is more interesting than lymph nodes (which may be small and difficult to find). However, for a patient’s prognosis, and thus for planning therapeutic options, the status of the lymph nodes is almost always more important than documenting a known primary tumor. Lymph nodes free of tumor may indicate a surgical cure, whereas tumor metastatic to lymph nodes signifies a worse prognosis and is often an indication for systemic chemotherapy or hormonal therapy. Treating fatty tissue with clearing agents facilitates finding small nodes but small nodes can also be found with careful sectioning and palpation.

Enlarged lymph nodes must be searched for diligently in all resections . Occasionally an occult primary carcinoma or an unsuspected lymphoma is discovered by finding an involved lymph node in a resection for benign disease.

If fewer than expected lymph nodes are found, the possible explanations include the following:

• Pathology factors: The prosector may not have found or sampled all of the lymph nodes in the specimen.

• Patient factors: Elderly patients tend to have fewer lymph nodes. Patients who have had prior surgery that transects lymphatics may have fewer lymph nodes. However, patients with rheumatologic diseases can have nodal lymphoid hyperplasia, making lymph nodes larger and easier to identify.

• Tumor factors: Some types of cancers (e.g., carcinomas with medullary features of the breast or medullary type carcinomas of the GI tract) are associated with nodal lymphoid hyperplasia making lymph nodes larger and easier to identify. Lymph nodes involved by metastases tend to be larger and more easily palpable.

• Treatment factors: Radiation and/or chemotherapy can reduce the number of lymph nodes.

• Surgical factors: The specimen may be small in size and/or may not include the appropriate tissue containing lymph nodes.

The pathologist should eliminate a poor gross examination as the reason for a small number of nodes in all cases. If only a few lymph nodes are found initially, it is of value to reexamine the specimen and to submit any additional tissue that may contain nodes for microscopic examination. A careful search should be documented in the report (e.g., “The axillary tail is thinly sectioned and palpated and all firm tissue is submitted for histologic examination.”). See “Lymph Nodes for Tumor Staging” for additional information on processing and reporting lymph nodes.

Margins

Margins are taken on all resections to document the presence or absence of tumor. Margin sections are taken in the area most likely to show involvement by tumor (i.e., at the closest approach of the tumor).

Orientation of margins for final diagnosis can be achieved by the following methods:

• Documentation of the site in the cassette key (e.g., “Cassette 3: Proximal sigmoid colon margin, perpendicular”).

• Use of colored inks to mark specific designated margins. The orientation should also be given in the cassette key to avoid mistaking artifactual ink for a true margin (e.g., “Cassette 2: Anterior margin [inked blue], perpendicular”).

There are two types of margins: en face and perpendicular to the plane of resection. The type of margin must be specified in the dictation, as this will determine whether or not a margin should be considered positive. For some specimens (e.g., skin excisions) a combination of en face and perpendicular margins may be useful.

En face margins (shave, parallel, orange peel; Fig. 2.1 ): The margin is taken parallel to the plane of resection. This has been likened to removing an orange peel and examining the flattened surface.

Advantages

• 10–100-fold greater surface area can be examined than when sections are taken in a perpendicular plane.

• An entire anatomic structure can be evaluated (e.g., the complete circumference of a bronchus or ureter).

Disadvantages

• The exact distance of the tumor from the margin cannot be measured in most cases. Tumor can be reported to be within the width of the section to the margin (usually within 0.2–0.3 cm). If the tissue is carefully oriented and embedded such that the first section cut is the tissue at the margin, it may be possible to determine if the margin is positive.

• This type of margin must be specified in the dictation, as, unlike perpendicular margins, any tumor in the section is considered to be “at the margin” regardless of the location of ink on the slide (e.g., the tumor may not be at ink at the periphery of the tissue section but could be at the margin in the center of the section).

• Pathologists are accustomed to evaluating perpendicular margins in the majority of specimens (except for bronchial and ureteral margins) and may not be aware that the margin is en face.

• Cautery artifact is often present and can make interpretation difficult.

The orientation of an en face margin as it is embedded for histologic sections either for frozen sections or in a paraffin block for permanent sections is important for tumors for which a narrow rim of normal tissue would be considered to be a negative margin. The tissue may be embedded so that the first cut section is the true margin. If the opposite face is cut first, and tumor is present, then deeper sections may be obtained, or the tissue re-embedded in the opposite orientation, to evaluate the “true” margin. If specific orientation is important, the side of the tissue to be cut first should be inked orange and a note written on the histology log sheet (e.g., “orange inked side to be cut first”). It is also advisable to speak to a histotechnologist about the case. It cannot be assumed that the orientation of the tissue in the cassette will be the same as the orientation of the embedded tissue.

Perpendicular margins ( Fig. 2.1 ): The margin is taken perpendicular to the plane of resection.

Advantages

• The exact distance of the tumor from the margin can be determined. Perpendicular margins are recommended when a small rim (e.g., less than 0.2 cm) of uninvolved tissue would be considered a negative margin.

• The majority of pathologists are familiar with interpreting this type of margin.

Disadvantages

• Very little tissue at the margin is actually sampled in large resections.

Inks

There are tissue marking dyes specifically marketed for medical use (e.g., Cancer Diagnostics, Inc. [CDI], Morrisville, NC; Bradley Products, Davidson Marking System [DMS], Bloomington, MN). However, there are less expensive alternatives such as India ink and acrylic ink marketed for artwork. ^, It is important to note that some inks can change or lose color after tissue processing, decalcification, or processing for immunohistochemistry. Therefore, it is important for pathologists to be aware of the properties of the inks used in their institution and to evaluate colors on routinely processed tissue. The colors most readily identified by pathologists are black, green, red, and blue. Typical problems are distinguishing yellow from orange, orange from red, and violet from blue. In a thin section on a glass slide, the predominant color that is easily identified grossly can be intermingled with microscopic contamination by other colors, making margin identification difficult or ambiguous. Therefore, the macroscopic color of the margin should be included in the cassette key so as to not solely rely on microscopic identification.

Method: The outer surface of the specimen should be relatively clean and dry. Ink may be applied with a gauze pad, a cotton swab, or by immersing the entire specimen into a container of ink. After applying the ink, some pathologists apply dilute acetic acid (5%) or methanol, which may act as mordants and help fix the ink to the tissue and prevent it from dissolving in formalin. However, good results can be obtained by ensuring the inked surface is allowed to dry or blotted to prevent smearing ink on interior surfaces. The best technique may depend on the specific types of inks used by the institution.

When multiple colors of inks are used for orientation, it is helpful to establish an institutional system for relating colors to orientation (e.g., yellow is always used for “lateral,” black for “posterior,” etc.).

Margins are sometimes stapled. The staples typically cannot be removed without shredding the tissue. The staple line can be carefully cut away as close as possible to the staples. The tissue directly underneath the staples is inked and the next closest tissue taken as the margin. Sections that contain staples should never be submitted for histologic processing, as the staples will damage or destroy microtome blades and the tissue adjacent to the staple cannot be cut for examination. One possible exception is evaluation of the staple line of a lung wedge resection during intraoperative consultation, because some staples can be removed after tissue is frozen (see Lindberg MR, Lung: Margins, Diagnostic Pathology: Intraoperative Consultation, Ed: SC Lester, Elsevier, 2nd ed., 2018).

Multiple Lesions

Occasionally multiple gross lesions are found in a specimen. It is important for both diagnosis and prognosis to determine if these lesions represent (1) the same lesion with a microscopic interconnection between the two gross lesions; (2) a primary tumor and a metastasis; or (3) two independent lesions. The distance between lesions is recorded and each lesion is sampled separately, and special studies taken as indicated. Always submit a section of tissue between two (or more) lesions to evaluate whether they are truly separate or interconnected.

If the lesions are within 2 cm of each other, it may be possible to take a longitudinal section that will include both lesions and the intervening tissue.

Missing Specimens

Specimens may be missing because the specimen was never accessioned, because the specimen container was received and was empty, or the specimen cannot be located after specimen accessioning. Missing specimens may have medicolegal implications in some cases. There may be an institutional requirement to file an incident report, and it may be prudent to contact a risk management department. Some states may also have requirements to report this type of occurrence.

Gross Descriptions

A good gross description has the following qualities:

• Succinct and to the point. The important information can usually be captured in a few sentences. Long, rambling descriptions are often poor because important information is buried in, or replaced by, irrelevant details.

• Good organization. Information is easily overlooked if it is not readily accessible and in the correct anticipated location.

• Adequate dissection. A specimen cannot be described accurately until after it has been completely dissected and examined. Initial impressions often change after a thorough examination. Important findings and measurements can be recorded in a notebook to aid in dictation after the specimen has been dissected. This practice also provides a backup gross description if a transcription is lost.

• Standardization. Standardization minimizes the risk of omission of important information. Creative dictations should be reserved for the very unusual or complicated specimen. Sample dictations for all large specimens are included in individual chapters.

• Diagrams. Diagrams of complicated specimens are helpful to show the site of tissue blocks. Photocopies and photographs have also been used for this purpose. ^{, ,}

Formatting the Gross Description

Even the most complex resections (e.g., extrapleural pneumonectomies, complex hemipelvectomies with multiple organs, Whipple pancreaticoduodenectomies, “living autopsies”) can be clearly described and sampled by approaching the specimen systematically.

There are six components to a gross description:

• 1.

  The first part documents the patient’s name, the medical record number, the specimen label, whether the specimen was received fresh or in a type of fixative, and anatomic structures present in the specimen (with dimensions and weight as appropriate).

  The orientation of margins and relationship to inks marking the margins is described.

• 2.

  The second part begins the description of the main pathologic findings that caused the specimen to be resected (type of lesion, size, relationship to normal structures and margins, etc.).

• 3.

  The third part describes any additional pathologic lesions not described in the second part (e.g., incidental polyps, a second smaller lesion, diverticula).

• 4.

  The fourth part describes any other normal structures not conveniently fit into the first sentence (e.g., length and diameter of ureters from a bladder resection).

• 5.

  The fifth part lists frozen sections, photographs, radiographs, and any other special studies that were done. The margins are identified according to ink color and whether they are en face or perpendicular.

• 6.

  The sixth part is a list of the cassettes and the types of tissue sampled.