Taking a Biopsy

Clinical Management of the Cancer Patient

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Taking a Biopsy

Ronald F. Carter, DVM, PhD,* and Victor Edwin Oswald Valli, DVM, PhDt

In this article we will review for the private practitioner the process of biopsy for cytologic and histologic study. We will emphasize a logical approach and critical elements of technique which together ensure ·a satisfactory interaction with pathologists. In accordance with the subject of this issue, we will emphasize biopsy of suspected malignancies.

THE ROLE OF THE BIOPSY IN THE DIAGNOSTIC APPROACH A biopsy is tissue obtained from a living animal by some method of surgical removal, such as excision, aspiration, imprint, scraping, or wash. Although sampling peripheral blood for cellular or biochemical studies constitutes a biopsy, we will not consider such tests here. Indications A biopsy is obtained to (1) diagnose disease, (2) monitor response to therapy, or (3) obtain living cells for studies such as tissue culture. For most practical purposes, a biopsy is obtained for cytologic or histologic study intended to provide a diagnosis for a clinical problem. Another reason related to diagnosis of disease is the selection of tissue for bacterial or viral culture. Repetitive biopsies of specific tissues under treatment are occasionally performed. Examples would be resections of recurring masses, repeated synovial taps for joint disease, and sequential bone marrow aspirations. More rarely, biopsies are obtained for the specific purpose of obtaining living cells for study. justification of Surgical Intervention. As with any intervention, it is implicit that potential benefit to the patient should exceed potential risk. *Postdoctoral Fellow, Department of Pathology, University of Toronto, Toronto, Ontario, Canada t Diplomate, American College of Veterinary Pathologists; Dean, University of Illinois College of Veterinary Medicine, Urbana, Illinois Veterinary Clinics of North America: Small Animal Practice-Val. 20, No. 4, July 1990

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The decision to obtain a biopsy therefore depends in part upon an estimate of risk. The risks involved vary according to the severity of surgical intervention and anesthetic restraint required. For techniques such as fineneedle aspiration, risks may be very slight. In contrast, an exploratory laparotomy or thoracotomy of an aged and compromised patient may represent a significant risk of death. Biopsies obtained by laparoscopy or endoscopy may be reasonable alternatives to more invasive procedures despite the risk of diagnostic failure due to a smaller and possibly less representative specimen. While an estimate of risk may ultimately determine whether a biopsy should be taken from an animal, it is not the sole consideration. Estimate of risk alone is inadequate to determine the type of biopsy or technique to be used. Certain types of biopsy may result in more definitive and/or timely results. The least risky procedure is frequently not the most informative. Thus, the practitioner has to estimate the likelihood of obtaining the definitive answer versus the risk resulting from the necessary procedure. One should always attempt the most direct assay of the suspected lesion ppssible under the circumstances. A common approach is to investigate the nature of a suspicious lesion by cytology, which is rather noninvasive and provides a rapid diagnosis; if the findings are uncertain or indicate a serious lesion, follow-up excisional biopsy and confirmation by histologic interpretation is justified. Role of the Biopsy in the Diagnostic Plan. Biopsies should not be obtained unless there is a clear expectation of informative result. Although almost any tissue can be biopsied, biopsies provide specific information only about very localized areas of tissue; the surgical action required to obtain a biopsy may even preclude the simultaneous biopsy of a number of tissues while failing to provide information about those other tissues. Biopsies are unique in their ability to provide information about the living patient, because tissue is actually obtained for direct study. Biopsies are best used as the final test which confirms a diagnosis already suggested by history, clinical picture, panel tests, and/or ancillary diagnostic techniques. Biopsies performed as part of a survey based upon poorly formed clinical suspicions will often result in unnecessary injury with little diagnostic benefit. Relationship to Clinical Pathology and Autopsy. It is useful to review the relationship among clinical pathology (e. g., hematology and serum biochemistry), biopsy, and autopsy. Hematology constitutes a specific test for the hemopoietic system and also provides information about systemic disease and response. Serum biochemical panels are carefully constructed to provide surveys of systemic response as well as indications of specific organ function or injury. Biopsies provide premortem morphologic evidence of tissue function and disease. Autopsy provides a postmortem opportunity to selectively or exhaustively study all tissues for morphologic evidence of tissue function and disease. Autopsy will not provide information useful for the management of the patient in question, but it is essential for monitoring the accuracy of the diagnostic approach and the (lack of) effectiveness of clinical management. Summary. The decision to obtain a biopsy is reached after the history,

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physical examination, and noninvasive tests suggest a specific list of diseases related to an identified tissue. Undertaking biopsy requires that the risk of surgical intervention is justified by the benefit of resulting information. The choice of biopsy technique and specimen preparation should maximize the diagnostic value of the biopsy while minimizing the risk. Cytopathology can be used as a safe screening technique to provide rapid diagnoses or indicate the need for excisional biopsy and histopathology. For many diseases, biopsy represents the decisive diagnostic approach for the living patient. Autopsy, on the other hand, provides the ultimate assessment of the adequacy of diagnosis and management.

COMPONENTS OF THE BIOPSY PROCEDURE The complete process of taking a biopsy involves many steps (Fig. 1). Throughout, there are two paramount considerations. There must be constant maintenance of correct patient and specimen identification, and the correct diagnostic interpretation must be returned to the practitioner. Failure in these aims compromises the entire procedure. Biopsies can be classified by the nature of tissue sampled (Table 1), which in turn reflects the diagnostic dilemma facing the practitioner. One may sample fluids by aspiration (e.g., ocular, pleurallpericardial, synovial, peritoneal, cerebrospinal, urine, or cystic fluids, and lung or prostatic washes). One may sample tissues by fine-needle aspiration, even wellorganized connective tissue. However, surgical excision of tissue is still the most common method of biopsy, applicable to almost all tissues except fluids. The method of biopsy determines the type of laboratory processing for routine evaluation (see Table 1). The most common division is into cytologic specimens (which are examined without prior sectioning) and histologic specimens (which involve fixation, paraffin embedment, and sectioning before evaluation). Fluids and fine-needle aspirations are processed for cytology; solid tissues obtained by excision can provide cytologic specimens by making imprints or scrapings on slides (often useful for quickly assessing the adequacy of the biopsy and obtaining a provisional diagnosis), but are ultimately examined as histologic sections to obtain the most information. Not covered in detail in this review are the other types of diagnostic tests that can be performed on biopsies. Outlines of appropriate procedures for immunohistochemistry, electron microscopy, and other tests are summarized in Table 2. Techniques

Important points for taking biopsies and preparing specimens for submission are summarized in Figures 2 and 3. Diagnostic Assessment. The initial step in the biopsy procedure consists of an evaluation of history, signalment, and physical findings, which leads to a problem list and associated diagnostic plans. After ancillary testing where necessary, the clinician forms a list of possible causes for the problems identified. Biopsy may be indicated as a logical development of the

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BIOPSY

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PREPARATION & FIXING

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staining sectioning embedment Figure l. Flow chart of biopsy procedure.

IDENTIFICATION

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Table I. Types of Biopsies for Morphologic Study TISSUE TYPE

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SPECIMEN TYPE

PREPARATIOI\

Air-dried Film on Slide Direct/ Aspirate ~ Concentrated - - Wet-fixed Film on Slide (Optional) ....--"Air-dried Imprint on Slide~ Excisional or~ Wet-fixed Imprint on Slide~ Tru-Cut ~ (Optional) Biopsy Formalin Fixed, Paraffin~ Section

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ROUTINE STAIN

Wright's Papanicolaou Wright's Papanicolaou Hematoxylin & Eosin

diagnostic plans, and the appropriate biopsy procedure should be selected through evaluation of the type oflesion, the risk associated with the biopsy, and the likelihood of obtaining a definitive answer. Biopsy. The biopsy should be representative of the lesion. Fine-needle aspiration should be done in a methodical spraying motion, with the lesion immobilized by one hand, to ensure representative sampling of the lesion. Aspiration should be stopped as soon as there is either sufficient material obtained or dilution of the intended sample by frank blood. When resection is performed, the biopsy may consist of part of the lesion or the entire lesion as part of the approach to therapy. Resection of a mass should be represented by a specimen which consists of the normal tissue surrounding the mass, the interface of the mass and the normal tissue, and the center of the mass. If an abscessed, necrotic, or cystic lesion is identified, the biopsy should consist of viable peripheral tissue rather than the disintegrating center. Surgical removal should disrupt the existing morphology of the tissue as little as possible. When fine-needle aspiration is complete, suction pressure should be released before the needle is withdrawn from the lesion, so that the aspirated material remains in the hub of the needle and is not explosively released into the syringe by inrushing air. The needle is then detached from the hub, air is drawn into the syringe, and the needle is replaced preparatory to expelling the sample onto microscope slides. For histology, excessive surgical manipulation, crushing, and congestion are common artifacts of surgical procedure which cause major difficulties in interpretation (Fig. 4). If imprints are made, care should be taken not to crush the tissue while making the imprints (a common error with Tru-Cut biopsies). Fixation. The material obtained by biopsy should be immediately fixed, that is, processed in a manner which ensures conservation of morphologic detail over time and throughout subsequent processing (see Tables 1 and 2). Unfixed or poorly fixed cells lose morphologic detail. They are also difficult to prepare for examination because they may undergo lysis, fail to adhere to slides, or fail to take up stains properly. Delayed or improper fixation of a specimen can render interpretation of subtle, or even otherwise obvious, lesions difficult. Cells with high metabolic rates, such as gut epithelial cells and hemolymphatic cells, will start changing in morphologic

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Table 2. Outline of Specimens Required for Tests on Biopsies* DIAGNOSTIC TEST

POSSIBLE SPECIMENS

METHOD OF PREPAR"'TION

Cytology

Aspirated fluids or tissues, See Table l. Air-dried smears exudates, imprints, washes are routine. Assess (eg, bronchoalveolar lavage), requirement for scrapings if poor exfoliation concentration by initial quick stain

Histology

Any solid tissue, or any cells See Table l. Formalin fixative is routine; other fixatives which can be centrifuged to may be preferred for a pellet specific tissues, eg, B5 for bone marrow

Electron microscopy

Any solid tissue, or any cells Glutaraldehyde-Os04 fixation is routine; paraformaldehyde which can be centrifuged to a pellet may be best for immunoelectron microscopy. Formalin adequate for some purposes

Immunohistochemistry, eg, skin, autoimmune diseases

Any suspension of cells or solid tissue

Bacteriology, mycology, virology

Refrigerated transport and Any solid or fluid specimen holding until samples which can be collected cultured. Freezing aseptically. Solid tissue acceptable but room specimens should be large enough that cultures can be temperature to be avoided initiated from interior sites specifically

Cytochemistry & histochemistry, eg, storage diseases, leukemias

Cell suspensions or solid tissues

Submission of absolutely fresh specimens best. Transport in liquid nitrogen or specialized transport media (eg, Michel's media for skin samples) may be appropriate. Tests done on smears, frozen sections, or fixed tissue

Absolutely fresh tissues best. Other transport methods may be possible

*The first procedure should always be to contact the laboratory for exact requirements for a specific test.

detail within minutes of cardiovascular compromise (Fig. 5). Fixation therefore should be initiated rapidly and should be complete before further tissue processing. Cytologic specimens are usually prepared by quickly making air-dried smears (see Fig. 2) or imprints (see Fig. 3). Imprints are best made by gently rolling a freshly cut face of tissue across the slide, after the tissue face has been gently blotted dry with paper gauze. Fine-needle aspirates can be prepared as smears (or films) and crushes (Fig. 6, A and B). Crush preparations, made by placing the aspirated material between a slide and a coverslip and drawing the two apart, often cause significant loss of

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release vacuum before leaving tissue remove needle and reload syringe with air

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propel tissue out of needle hub using air in syringe Essential elements of Technique: 1.) 22 gauge needle, 10 ml syringe 2.) Accurate sampling of lesion; no dilution by bleeding 3.) Sample retained in hub of needle, not lost in syringe 4.) Rapid preparation of slides directly from aspirate 5.) Smear technique used to spread slides spread tissue out behind leadir.g edge of slide 6.) Rapid fixation by air drying or wet fixation (spray is best). 7.) Transport at room temperature in protected package air dry and/or wet fix rapidly

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Figure 2. Preparation of smears from fine-needle aspirates.

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obtain biopsy tissue without comprising morphology slice into 2 to 3 mm thick sections in appropriate orientation

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Essential elements of imprint technique: 1.) Gently cut fresh tissue face and blot off excess fluid 2.) Hold in one hand and gently roll slide over tissue face without creating a suction effect which rips cells 3.) Rapidly air-dry or wet fix (spray is best) 4.) Prepare only a few imprints from each tissue face

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Essential elements of formalin fixation: 1.) Gently handled fresh tissue 2.) Abundant volume of fresh, buffered 3.7% formalin at room temperature in secure specimen bottle 3.) Distance formalin required to penetrate to desired lesion < 3 mm 4.) Fix at least 24 hrs before processing regardless of sample size 5.) Avoid freezing before embedding

Figure 3. Preparation of imprints and fixed histologic specimens.

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Figure 4. Histologic section of laryngeal biopsy from a cat . Edge of tissue with crush artifact in fresh specimen . The irregular dark streaks are ruptured nuclei. 1-1 & E; bar = lO fJ.m.

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Figure 5. A, Histologic section of mediastinal mass from a cat with immunoblastic lymphoma. E arly autolysis is represented by cytoplasmic separation and contraction, and by irregular nuclear membranes and chromatin clearing. H & E ; bar = lO J.Lm. Illustration continued on opposite page

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Figure 5 (Continued). B, Histologic section of a mediastinal mass from a cat. More extensive autolysis is represented by ruptured nuclear membranes and fragmentation of chromatin. Cytoplasmic separation is again present. Stromal nuclei, in comparison, maintain a more normal appearance , except for vesiculation and peripheralized chromatin (arrow). The section is also too thick, obscuring cellular details. H & E ; bar = 10 fLm .

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Figure 5 (Continued). C, Histologic section of liver from a cat. Autopsy specimen. Subcapsular tissue has undergone chromatin lysis and cytoplasmic degradation due to effects of bile leakage prior to tissue fixation. Edge effects may also be caused by desiccation. H & E ; bar = 100 J.Lm, or 0.1 mm.

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Figure 6. A, Smear preparation; fine-needle aspirate of enlarged mesenteric lymph node from a dog. Wright's stain. Cluster of cells containing benign small lymphocytes intermixed with large epithelial cells. The epithelial cells have markedly enlarged, hyperchromatic nuclei, cribriform chromatin pattern, and huge nucleoli (arrow). Diagnosis of metastatic carcinoma was based primarily upon these cellular criteria. Cellular details and associations are well preserved. Bar = 10 j-t.m. Illustration continued on following page

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F igure 6 (Continued). B, Crush preparation; same specimen as in A . Wright's stain. Cellular membranes are ruptured. Many nuclei have been ruptured against tissue fibrils, causing long strands of chromatin to form. Crushing action also disrupts cell associations. Bar = 10 f.Lm.

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Figure 6 (Continued). C, Histologic section of pinch biopsy from stomach of same dog as in A and B. Proper technique has preserved architectural and cellular details. Isolated infiltrating tumor cells are present in the mucosa (arrow). H & E; bar = 100 fLm, or 0.1 mm.

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Figure 6 (Continued). D, Higher-power view of same section as in C . Diagnosis of gastric adenocarcinoma was based on presence of signet ring cells (A), with large secretion vacuoles representing abnormal cytoplasmic function (secretion) and form (compression of nucleus to edge of cell). Nuclear changes also associated with malignancy include increased nuclear and nucleolar size. Benign residual acinar cells (B) demonstrate normal cellular differentiation. H & E ; bar = 10 fJ-m.

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morphologic detail due to tearing and rupturing of cells. Smears are much less disruptive and provide better specimens, despite the fact that they may appear to inadequately spread the cells. Scrapings of particularly hard tissues can still be smeared onto slides with excellent preservation of detail. Aspirates of fluids may require preparation of direct and concentrated specimens in order to provide enough cells on a slide to be informative. Submission of excess fluid in tubes along with the slides may be useful to obtain cell counts or biochemical assays, but such fluids are useless for morphologic study because of autolysis of cells and bacterial overgrowth during transport. Bacterial overgrowth may be recognized by the presence of abundant large colonies of bacteria without any 'evidence of bacterial phagocytosis by macrophages or neutrophils. Fixation by air-drying is simple and effective, but the drying action must be rapid (placing fresh films or imprints in front of a srnall table-top fan works well). Air-dried specimens are then usually stained with Wright's stain, which will give good nuclear and cytoplasmic detail for single cells and cells in small clumps or clusters (Figs. 6A and 7A). Epithelial tissues and specimens with large clumps of cells may benefit from preparation of additional wet-fixed specimens. Freshly made specimens are sprayed with an aerosol fixative within 2 to 3 seconds of spreading the cells. Wet-fixed specimens are usually prepared with Papanicolaou's stain (Fig. 7B), which has several advantages over Wright's for the examination of thick clumps of cells in general as well as epithelial cells specifically. Because this stain clearly shows nucleolar morphology and also results in a more transparent staining of cytoplasm, nuclear morphology of cells in large clumps can be evaluated very well. The stain also identifies cornified epithelial cells by a characteristic orange reaction with keratin; this property is especially useful when carcinoma is suspected. Histologic specimens should be sliced immediately into 2 to 3 rnm thick slices. At this time, imprints can be made. The slices of tissue are placed into an abundant volume of fresh neutral buffered 3. 7% formalin. Although formalin penetrates specimens very quickly, it actually takes about 24 hours for formalin to adequately crosslink between molecules in the tissue and therefore fix morphologic detail. The 24 hours required for this action is independent of the size of the biopsy material. It is a common misconception that small specimens such as Tru-Cut biopsies can be quickly processed after only a few hours in formalin. Such an approach is destined to give inferior results (Fig. 8). The opposite is also true: Excessive fixation in formalin may result in overly fixed tissue which may be resistant to special procedures such as immunohistochemistry. Submission and Mailing. Once the biopsies are obtained and prepared for submission, the specimens should be clearly and permanently labeled so as to identify at least the owner, animal, and tissue. Labeling by waterproof and alcohol-proof marker pen, or thick pencil, is preferred. Labeling by ballpoint pen is often rendered unreadable, for example, by leakage of formalin from specimen bottles, or processing of slides through the solvents required for staining. Bottles should have adhesive labels. Slides should be labeled directly on a frosted end. Adhesive tape or other

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0 Figure 7. A , Air-dried imprint, Wright"s stain, of skin mass from a dog. Larger cells have prominent granulation (actually purple, or metachromatic, with Wright's stain) that occasionally obscures nuclear detail. Diagnosis of mast cell tumor was based on abnormal numbet· and size of mast cells in a specimen of canine skin (i.e. , appropriate history). Smaller cells are benign lymphocytes. Bar = 10 fLm.

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Figure 7 (Continued). B, We t-fixed imprint of same specimen as in A, prepared with Papanicolaou's stain. Cytoplasmic granules are not specifically stained ; therefore, nuclear detail and cellular associations in cluste rs are more apparent. Prominent nucleoli (arrow), an important prognostic feature in the evaluation of mast cell tumors, are readily apparent. Bar = 10 f.Lm .

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Figure 7 (Continued). C. Histologic section of lesion from which imprints in A and B were obtained. A dense cluster of hyperchromatic cells is apparent in the superficial dermis. Diagnosis of mast cell tumor was based upon alteration of architectural pattern and specific identification of cytoplasmic granules at higher power. H & E ; bar = 50 ,_.,m.

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Figure 8. Histologic section of pinch biopsy of nasal turbinate from a cat. Despite the small sample, incomplete fixation is indicated by bland homogeneous nuclei and cytoplasmic separation. Less apparent are tissue compression and increased section thickness, made more like ly because of difficulties associated with processing of poorly fixed tissue. H & E; bar = 10 j.Lm.

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sticky labels increase the thickness of slides and interfere with the action of some automatic stainers, and also will take up stain when possible, making the labels unreadable. A submission form appropriate for the intended laboratory should be filled out, supplying identification as well as additional relevant data such as the clinic, clinician, date, species, age, summary of clinical history and physical findings, description of tissue taken in the biopsy (including size, site, attachment and other significant properties), and recent treatment. Of great importance, especially for cytologic specimens, is a list of diagnostic rule-outs or suspected conditions. Such information is critical for the proper evaluation of the specimen by the pathologist, because many findings ar.e interpreted in relation to the clinical data supplied; providing clinical data helps ensure that the resulting information includes interpretation and inference rather than just mort>hologic description. The most important attributes of the chosen method of transport are minimal breakage, reliability, appropriate speed, and cost. No known method of packaging can guarantee safe passage by mail; breakage of protected slides and "unbreakable" plastic specimen vials, enclosed in padding in hard cardboard mailing tubes, exceeded 10% for a recent series of biopsies mailed to our institution. Breakage seems to be minimized when specimens are placed in styrofoam chips or bubble pack wraps in an overly large cardboard box or mailing tube. History sheets should be placed in separate plastic bags so that if leakage of formalin or other fixatives occurs there is no loss of information. Postal regulations regarding the mailing of biohazardous material should be followed. Freezing is another peril associated with transport of specimens. Despite the fixative effect of formalin, specimens which freeze while in formalin are marked by considerable artifact related to ice crystal formation once sectioned and stained. Similarly, cytology specimens which are refrigerated or frozen may suffer lysis of cells due to condensation of moisture on the specimen when returned to room temperature. Summary. The practitioner's aim is to provide the laboratory with a clearly identified and properly prepared sample, along with a clinical history, of the most direct and representative biopsy he can obtain. Manipulation of the tissue should be minimized. Preparation and fixation of the sample should be appropriate for the intended tests, so that there is preservation of cellular detail throughout the subsequent transport and laboratory procedures. Direct contact with the lab is appropriate if there is any doubt about the type of specimen required to confirm a specific diagnosis. The importance of submitting a clinical history cannot be overemphasized, because such information may be critical for a complete and proper evaluation of the lesion. Technical Laboratory Procedures

Aims of the Laboratory. When the laboratory receives the sample, it is processed through a standardized set of procedures designed to ensure confidential, reliable, timely, and accurate reporting of results. Laboratories work best when submitted specimens conform to a routine standard. A typical protocol consists of assigning a specimen number after logging in

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the data, labeling the specimens with the number, processing the specimens for evaluation, interpreting the specimens, preparing and mailing a report, and biiling. Processing methods vary for different types of specimens. Cytologic Specimens. Routine staining of air-dried specimens is a simple process, but it must be done well in order to resolve cellular details (see Figs. 6A and 7A). Wright's stain is done manually in batches or on automated equipment such as the Ames Hema-Tek instrument. Quick stains such as new methylene blue or other proprietary stains may be more easily performed in a practitioner's lab but generally result in inferior preparations. Papanicolaou's stain (see Fig. 7B) is more complex, requiring three different staining reagents as well as graded solvents. Coverslipping with a permanent mounting medium ensures protection of the specimen and optimal specimen resolution at lower magnifications. Histologic Specimens. Specimens are sent as slices of tissue fixed in neutral buffered 3. 7% formalin. A smaller portion may be selected, which is then loaded into a plastic cassette and placed in a processor which sequentially processes batches of cassettes through a graded series of alcohol and finally xylene solutions to remove all traces of water in the specimens. The final solvent must be miscible with the embedding medium chosen. This combination is often xylene as a solvent for embedment in paraffin wax. The specimens are then saturated with liquid paraffin wax, reoriented in the cassette for sectioning, and cooled so as to solidifY the paraffin. The purpose of embedment is to provide a supporting matrix throughout the specimen which resists the crushing action of sectioning. The cassettes with blocks of paraffin containing embedded specimens are then mounted on a microtome for sectioning. A microtome is an apparatus that advances the face of the tissue toward a metal knife edge at a very reproducible rate of advance and cutting speed. The cutting action produces a ribbon of sections of paraffin and embedded tissue. Routine section thickness is between 4 and 8 IJ.m; thickness of 6 IJ.m or less ensures a single layer of nuclei in the section and allows better resolution of detail in the section. Representative sections are picked up, floated on warm water to stretch out wrinkles and creases, and then placed onto slides and heated at about 50°C for a few hours to ensure that the section is dry and sticks to the slide during staining. The purpose of staining is to increase the visibility of tissue structures by routine bright field light microscopy. A combination of hematoxylin (a basophilic stain which renders nuclei blue) and eosin (an acidophilic stain which renders cytoplasm pink) is almost universally used. Other special stains may be required to detect specific substances or organisms. Again, stained specimens are coverslipped with a permanent mounting medium to provide protection and increased resolution of the specimen at lower magnifications. The quality of histologic processing directly affects limits of interpretation (Fig. 9). Embedding, sectioning, and staining must all be performed with appropriate care to ensure preservation and illustration of morphologic detail (see Figs. 6C, 6D, and 7C). Summary. The laboratory's primary concerns are confidentiality, reliability, speed, and accuracy. Proper preparation of routine specimens

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Figure 9. Histologic section of mediastinal mass from a cat. Artifacts caused by poor tissue processing include increased section thickness, irregular thickness due to nicks in microtome knife (arrow), and crushing and ripping of section due to dull knife . H & E ; bar = 100 fLm , or 0.1 mm.

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allows the laboratory to achieve these aims with the least difficulty and cost. The laboratory processes the specimens in a manner intended to render visible the morphologic cellular details required for interpretation. The laboratory cannot resurrect cellular details already lost because of inadequate specimens. Diagnostic Interpretation and Reporting

Interpretive Process. Despite variations in the interpretive process, common steps would include (1) recognition of the ·type of tissue; (2) recognition of abnormality; (3) when malignancy is considered, distinction of a benign -versus malignant lesion; (4) diagnosis; and (5) consideration of prognostic indicators, if any, present in the tissue. The basic philosophy of the pathologist, especially where malignancy is suspected, is to be cautiously accurate. Designating a lesion as benign when it is in fact malignant results in a delay of appropriate therapy, which may have serious consequences. Designatibn of a benign lesion as malignant may result in inappropriately severe therapy or euthanasia. The pathologist tries to report to the limit of what can be supported by interpretation, but if there is doubt about that limit, the report should err on the side of caution. Suggesting resampling or alternative types of biopsy is reasonable when the pathologist knows that the reported diagnosis is not definitive. Diagnostic caution often prompts the cytopathologist to suggest that findings support a specific diagnosis but that histology is required to confirm the findings. The cytopathologist and histopathologist perform similar protocols of interpretation, but there are some differences of approach made necessary by the differences in specimens. The cytopathologist does not generally have the benefit of an intact architectural pattern of the tissue. Specimens consist of only single cells or small groups of cells which have exfoliated out of the tissue, either through forceful encouragement (aspiration or scraping) or through looseness in relation to other cells (fluids and imprints). While imprints may have some correlation between cell distribution and the original architectural pattern of the tissue, no such correlations exist for aspirated cells. Therefore, the cytologist tries to glean as much information as possible from examination of the cells themselves. Guidelines of interpretation are formed on the basis of cellular morphology alone (see Figs. 6 and 7). In addition, the cytopathologist usually has a far smaller amount of sample to examine, and therefore accepts a greater likelihood that the sample is insufficient or unrepresentative. In these contexts, the cytopathologist is much more dependent upon submission of a good concise summary of the clinical history in order to make the most of what there is to study. In contrast, the histopathologist has the entire specimen. The histopathologist does not have to consider which cells might have exfoliated and which ones might not. The architectural pattern and overall arrangement of the lesion in comparison to surrounding tissues can be directly evaluated, in addition to cellular details. Guidelines of interpretation are formed on the basis of architectural pattern and cellular morphology (see Figs. 6 and 7).

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Tru-Cut and pinch biopsies excepted, the histopathologist is usually not as concerned about the adequacy or representative nature of the specimen. Criteria of Malignancy. The essential decision required in the diagnosis of malignancy is distinction of a dysplastic process (reaction to injury, inflammation, sepsis, or other cause of irritation) from a frankly anaplastic, or malignant, process. Often, the anaplastic nature of the tissue is so obvious that the decision can be made on morphologic criteria alone. Sometimes, however, the appearance of the lesion must be considered in relation to the absence of any detected predisposing cause. For example, septic rhinitis may cause reactive proliferation of sinusal mucosa, which is very difficult to differentiate from carcinoma. In such cases, the pathologist may recommend that the septic condition be treated, and that the lesion be resampled if persistent. Upon resampling, if the inflammation and sepsis were no longer present but severe dysplasia persisted, then it could be argued that a reactive process would be less likely and that the real nature of the lesion was a carcinoma which subsequently became septic and inflammatory. More rarely, the malignant nature of a morphologically benign lesion becomes evident only by clinical progression not predicted by biopsy. Criteria of malignancy are formulated to distinguish various clinically significant stages along the continuum of hyperplasia, dysplasia, and anaplasia. A general outline is given in Table 3, but criteria vary for different tissues. For example, nuclei of reactive benign fibroblasts will show an increase in overall nuclear size, a rounder shape, larger and more numerous nucleoli, greater variation in nuclear size (anisokaryosis) and basophilia, and Table 3. Criteria of Malignancy HISTOLOGY Tissue Level (Low-Power Examination) Local or distant invasion and obliteration of other tissues Loss or effacement of normal tissue architectural pattern Evidence of altered cell function, eg, inappropriate cell secretions Cellular Level (High-Power Examination): As for cytology CYTOLOGY Nuclei Abnormal variation in nuclear size or shape (anisokaryosis) Abnormally dense staining (hyperchromicity, related to ploidy) Abnormally large or multiple nucleoli (related to transcription and translation) General increase in nuclear size (related to ploidy) Abnormal mitotic rate (frequently increased), abnormally shaped mitotic figures Increased multinucleation, especially 3, 4, 5, or more nuclei per cell Nuclear crowding: abnormally close apposition of nuclear membranes Changes in chromatin organization Overall changes in nuclear shape Inappropriate nuclear differentiation compared to cytoplasmic differentiation Cytoplasm Abnormal variation in shape, size, or staining pattern Loss of differentiated characteristics ("undifferentiated") Inappropriate differentiation for tissue type (metaplasia) or nuclear differentiation Abnormally fragiJe, easily ruptured Loss of normal relationship to other cells or extracellular structures Abnormal pigment or other cellular product; abnormal cell function, eg, phagocytosis

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more frequent binucleate cells or mitotic figures compared to quiescent fibroblasts. Malignant fibroblast nuclei will show similar changes which are more excessive in degree, along with hyperchromicity (greater nuclear basophilia directly related to increased ploidy), more mitotic figures, and bizarre multinucleated cells. In contrast, in bone marrow and lymph node, it is normal for cells to divide and differentiate into cells of different nuclear appearance; a homogeneous population of cells is highly suspicious. Thus, variation in nuclear appearance is not always a hallmark of anaplasia. Again, cytologic criteria are entirely based upon the morphology of cells (see Figs. 6 and 7). Cytologists pay great attention to nuclear morphology in order to differentiate dysplasia from malignancy. Cytoplasmic details are. primarily used to identifY the tissue type rather than its intentions. The organization of small clumps or clusters of cells, as well as cytoplasmic borders, staining, and products, are important indicators of tissue type. Histologic criteria of malignancy are primarily based upon disruption of normal relationships of tissue types as shown by architectural pattern (see Figs. 6 and 7). Local or distant invasion of other tissues, or obliteration of normal structure-function relationships in the lesion and surrounding tissues, represents visible evidence of malignant properties. Delineation of the limits of the lesion, and the adequacy of the excision, are specific indications for histologic study related directly to study of architectural pattern. In addition, the histopathologist also has cellular morphology to further provide support for the eventual diagnosis. Cellular morphology is used to provide the specific diagnosis of tissue type and may b.e the basis for detection of small foci of cancer such as carcinoma in situ. One of the great difficulties of cancer diagnosis is that malignant cells frequently lose the characteristics of differentiation which allow classification of benign tissue types. This leads to classification based upon more specific indicators than cellular morphology, such as specific cell products (for example, the DOPA stain for melanin in malignant melanomas), expression of specific antigens (immunohistochemistry for intermediate filament expression to differentiate carcinomas from sarcomas), or clonal genetic rearrangements (immunoglobulin gene rearrangement to differentiate Bcell from T-cell lymphomas and lymphoid leukemias). Studies of cellular products or antigen expression are complicated because there may be aberrant or apparently meaningless expression of characteristics which are quite definitive for benign tissues. Further, there is currently little prognostic benefit from such characterizations despite the satisfaction of a more accurate diagnosis. Eventually, however, more accurate delineations of clinical entities will result in more effective treatments for each entity. Thus, it is appropriate to try to be as specific in a diagnosis as possible. Reporting. A complete written report should be in a format consistent for the laboratory, understandable to the practitioner, and in conformance with expected standards of identification, description, diagnosis, and prognosis where possible. The report is often founded upon specific cornerstones. For cytology, there is usually a description of (l) background (noncellular) material; (2) cells; (3) abnormal cells, cellular products, or organisms; (4) morphologic

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diagnosis; and (5) etiologic diagnosis and/or prognosis if possible. For histology, there is usually des9ription of (1) macroscopic characteristics of the lesion; (2) relationship of lesion to surrounding tissues; (3) architectural pattern of lesion and benign tissues; (4) cellular morphology; (5) abnormal cell populations, cellular products, or organisms; (6) morphologic diagnosis; and (7) etiologic diagnosis and/or prognosis where possible. Reports often consist only of statements of morphologic and/or etiologic diagnoses. Despite the expediency, this is deficient. First, assuming the diagnosis is correct, there is still a considerable loss of information to the practitioner. Second, if the interpretation is incorrect, there is no reason for anyone else to suspect this on the basis of the report. Complete reports provide a description of the lesion as well as the diagnosis, so that any other knowledgeable diagnostician can evaluate the lesion somewhat independently. Lastly, if follow-up biopsies are taken, it is more difficult to evaluate the progression or resolution of the lesion based only on diagnoses. Although the pathologist can quite correctly limit the report to diagnosis and interpretation, it may be more useful to the practitioner if inferences are made. For example, simply reporting a diagnosis of mammary carcinoma in a dog may not be nearly as helpful as suggesting a prognosis related to species, cell type, extent of invasion, and presence or absence of inflammatory reaction. However, the practitioner must accept that an inference is subject to uncertainty. Implementation of the Reported Information. The practitioner arrives at a diagnosis and plan of action by considering the reported information along with other results and clinical impressions. The practitioner receives the report some time after submitting the specimens; in the meantime, there may have been further progression of the lesion, and results from other tests received. The aim is to synthesize the incoming data into a list of findings which support a specific diagnosis. Verbal discussion with the pathologist may be very helpful, especially where terminology in the report is obscure to the practitioner. In most cases, the revised diagnosis will be substantially the same as the provisional diagnosis which justified the testing. With the correct diagnosis, appropriate therapy can be selected. Further testing may be used to monitor the response to therapy. As always, an autopsy is the final chance to evaluate the quality of case management. Summary. The pathologist's aim is to provide a cautious, accurate diagnosis for the lesion under consideration. There are specific steps of specimen evaluation common to a wide variety of specimens and tissues. Limitations of interpretation are greater for cytopathology than for histopathology, primarily because the architectural pattern of tissue differentiation is rarely assessable. Despite this, cytopathology and histopathology have complimentary roles in the assessment of biopsies (Table 4). Aberrant cellular differentiation and behavior are morphologic criteria of malignancy. Criteria for dysplasia and anaplasia vary by cell type, but extreme anaplasia complicates designation of tissue type because of aberrant expression of differentiating characteristics. A complete report consists of identification, description, diagnosis, and prognosis where possible, for the specimens submitted. The practitioner should contact the pathologist directly for clarification

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Table 4. Morphology: Advantages and Limitations of Cytology and Histology CYTOLOGY

Advantages Safe, relatively noninvasive; can sample without surgical excision Rapid and simple Sample can be prepared, stained, and examined by practitioner if desired Applicable to a wide variety of different types of specimens and diagnostic techniques Limitations Greater possibility of nonrepresentative sample bec.ause (a) essentially blind sampling of lesion (imprints excepted); therefore localization may be imperfect; (b) very little tissue obtained; and (c) only certain cells may exfoliate Sample more vulnerable to destruction and aging effects until stained and coverslipped; then, no more tissue available for other special stains Architectural pattern of tissue not available, therefore greater limitation in interpretation

HISTOLOGY

Advantages Actual removal of desired tissue Can prepare cytologic specimens from histologic specimens Histologic preparations give architectural pattern and cell morphology, therefore definitive interpretation; direct assessment of limits of tumor infiltration possible Usually tissue is still left over after sections prepared, therefore abundant material left for special techniques Specimens less vulnerable to destruction, except freezing or desiccation Limitations Excisional surgery required: (a) risk may be considerable; (b) excised tissue no longer functional; (c) not applicable to certain tissue types, especially functionally sensitive tissues and fluids Sample preparation complex and slow Practitioner cannot evaluate sample

of terminology or significance of the result. The report should also be evaluated for consistency with clinical progression of the case and results from other tests. eritical evaluation and implementation of results represent final roles of the practitioner in a cycle of collaboration initiated by the recognition of a clinical problem (see Fig. 1). On the basis of a confirmed or amended diagnosis, appropriate therapy can be instituted. Follow-up Table 5. Critical Requirements for Successful Biopsy Technique The Practitioner Appropriate diagnostic assessment, formulation of problem list The most direct and representative sample possible at acceptable risk level Contact with lab regarding specimen preparation if any doubt Appropriately gentle biopsy technique Proper specimen preparation Submission of properly labeled sample in protective packaging along with clinical history The Laboratory Maintenance of specimen identification and confidentiality Proper processing techniques Correct interpretation of lesion Proper report: identification, description, diagnosis, and prognosis if possible Timely reporting The Practitioner Collect all returning data and implement in revised case assessment, management Critical evaluation of report Contact lab directly if questions about interpretation or repeat samples

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biopsies and/or autopsy are· important for monitoring the effectiveness of case management.

CONCLUSIONS In our review of the process of taking a biopsy, the intent has been to show how proper diagnostic evaluation and technique can result in productive interaction with the laboratory and pathologist. The practitioner and the laboratory have different roles, and each must fulfill certain requirements (Table 5). Failure to follow appropriate procedures will result in unnecessary injury to patients, poor development of diagnostic acumen, delays in instituting appropriate therapy, and unjustified costs to the client. It is important that diagnosis be based upon the most direct assay possible of a properly evaluated lesion. Simple techniques of biopsy preparation for submission can then allow a proper evaluation of the lesion in the context of clinical findings. Pathologists attempt to be as diagnostically accurate as possible within limits of interpretation. Practitioners should critically assess the descriptive and diagnostic information in pathology reports and directly contact pathologists where questions still exist about the significance of results. One way for a practitioner to assess how well the biopsy procedure is functioning is to compare expected results with actual results. If only occasional discrepancies exist between tentative diagnostic rule-outs and confirmed diagnoses, then the implementation of diagnosis by biopsy is appropriate.

REFERENCES l. Frost JK: The Cell in Health and Disease: An Evaluation of Cellular Morphologic Expression

2. 3. 4. 5.

of Biologic Behavior. Monographs in Clinical Cytology, vol 2, ed 2. Basel, S Karger, 1986 Jacobs RM (ed): Diagnostic Cytology. Semin Vet Med Surg (Small Anim) 3(2), 1988 Koss LG, Woyke S, Olszewski W: Aspiration Biopsy: Cytologic Interpretation and Histologic Bases. New York, Ikagu-Shoin, 1984 Parry BW (ed): Clinical Pathology: Part I. Vet Clin North Am [Small Anim Pract] 19(4), 1989 Parry BW (ed): Clinical Pathology: Part II. Vet Clin North Am [Small Anim Pract] 19(5), 1989

Address reprint requests to V.E.O. Valli, DVM, PhD 3505 Veterinary Medical Basic Sciences Building College of Veterinary Medicine University of Illinois 2001 South Lincoln Avenue Urbana, IL 61801