Staci Beamer, MD, Dawn E. Jaroszewski, MD, Robert W. Viggiano, MD, and Maxwell L. Smith, MD
Optimal specimen handling is essential for the accurate interpretation of biopsies and cytologic preparations obtained in the course of evaluating the patient with lung disease.1-9 the limited number of sampling techniques available can be divided into three general categories: bronchoscopy, transthoracic needle core biopsy or aspiration, and surgical wedge biopsy of peripheral lung through a transthoracic approach.8’10-13
The focus of this chapter is on these techniques and the specimens There by obtained, with emphasis on how they should be prepared and handled in the laboratory. After an appropriate sample of adequate quality has been obtained, the addition of pertinent clinical data and radiologic information greatly increases the likelihood of a meaningful and accurate diagnosis.13-15 Even when the diagnostic goal is simply to rule out malignancy, the effect of other information may be substantial, especially when the sample is of marginal quality or size. In the case of diffuse nonneoplastic lung diseases (often referred to as interstitial lung diseases'), a reasonable amount of clinical and radiologic information is essential for accurate interpretation. Without such information, even the experienced lung pathologist may need to resort to a purely descriptive diagnosis.15
In this chapter, specimen characteristics and processing steps are presented for each of the common lung samples taken in the course of clinical evaluation for pulmonary disease. In addition, for each type of sample, the benefits and limitations are reviewed. Such a working knowledge of specimen handling for each procedure ensures the greatest likelihood of success in establishing a specific diagnosis and, in the end, a rational treatment plan. An overview of biopsy procedures and the specimens generated is presented in Table 3.1.
Specimens Obtained Through the Flexible Bronchoscope
The flexible bronchoscope was introduced in the United States in the late 1960s, after successful use in Japan.8 Despite several decades of experience with the rigid bronchoscope, the advent of the flexible bronchoscope (Fig. 3.1) allowed evaluation of the major conducting airways without use of general anesthesia and with less morbidity.8,16 Furthermore, the flexible instrument has the advantage of providing better access to more distal and obliquely branched airways. The rigid bronchoscope still has major uses in certain settings, mainly those in which the device’s larger bore is an advantage, but nowadays pulmonary endoscopy is dominated by the flexible bronchoscope.
Endobronchial Biopsy
Modern flexible bronchoscopes allow the operator to accurately visualize the structural integrity of the bronchial tree and its mucosal surfaces, commonly as far distal as the sixth-order bronchi17’18 (Fig. 3.2). Biopsy of visualized mucosal lesions most commonly is performed using cupped forceps (Fig. 3.3A) introduced through the flexible shaft of the bronchoscope.8 With this technique, the airway mucosa, lamina propria, and musculature are sampled with or without fragments of cartilage (see Fig. 3.3B). The closed forceps is extracted from the bronchoscope, and the biopsy is dislodged from the cupped ends of the device and placed in fixative or other solution (see later discussion). A sterile needle or fine-tipped forceps (Fig. 3.4) is useful for removing the delicate tissue specimen. The tissue specimens obtained in this way average 2 to 3 mm in greatest dimension. The lymphovascular network of the peribronchial sheath often is included in these samples, making it possible to identify metastatic disease when present in lymphatic or vascular channels (Fig. 3.5).
Table 3.1 Diagnostic Sampling Techniques, Specimens Obtained, and Common Analyses Performed
|
Sampling Technique |
Specimens/Common Analyses |
|
Sputum expectoration |
Cytologic smears and centrifuge preparations Fixed or air dried, then stained for cytopathologic examination Microbiologic cultures performed as indicated |
|
Bronchoscopy with: Washings |
Cytologic smears and centrifuge preparations Fixed or air dried, then stained for cytopathologic examination Microbiologic cultures performed as indicated |
|
Brushings |
Cytologic smears and centrifuge preparations Fixed or air dried, then stained for cytopathologic examination Microbiologic cultures performed as indicated |
|
Endobronchial biopsy |
Forceps tissue biopsy specimen, 2-3 mm in size Fixed and processed for histopathologic examination Microbiologic cultures and other testing performed as indicated |
|
Transbronchial biopsy |
Forceps tissue biopsy specimen, 2-3 mm in size Processed for histopathologic examination Microbiologic cultures and other testing performed as indicated |
|
Bronchoalveolar lavage |
Cytologic smears and centrifuge preparations Fixed or air dried, then stained for cytopathologic examination and biochemical analysis Microbiologic cultures and other testing performed as indicated |
|
Transbronchial fine-needle aspiration and endobronchial ultrasound |
Cytologic smears and centrifuge preparations Fixed or air dried, then stained for cytopathologic examination Microbiologic cultures and other testing performed as indicated |
|
Cryobiopsy |
Cryoprobe used to obtain multiple fragments of lung tissue, usually >5 mm each Fixed and processed for histopathologic assessment Useful for nodules and diffuse parenchymal lung disease |
|
Surgical "wedge" lung biopsy (either video assisted or open) |
Peripheral lung tissue sample (3-5 cm) including pleura and alveolar parenchyma Fixed and processed for histopathologic examination Microbiologic cultures and specialized testing performed as indicated |
|
Transthoracic needle core biopsy and aspiration |
Core biopsy fragment(s), cytologic smears, and centrifuge preparations Smears and cellular preparations: fixed or air dried, then stained for cytopathologic examination, with special stains for organisms and other specialized techniques as indicated Core tissue specimens: fixed and processed for histopathologic examination Microbiologic cultures and specialized assays performed as indicated |
|
Thoracentesis |
Cytologic centrifuge preparations Fixed or air dried, then stained for cytopathologic examination Microbiologic cultures, biochemical analysis, and specialized assays performed as indicated |
To avoid drying, specimens should be placed immediately into fixative solution or other transport medium. Immediate agitation of the samples in the solution vial helps to reexpand any crush artifact induced by the biopsy procedure, and, if done in a carrying medium, the supernatant can be aliquoted and sent for cytopathologic evaluation or special studies (e.g., immunocytochemistry studies, molecular genetic analysis). When these solutions are not available in the bronchoscopy suite or at the bedside, specimens can be placed in a closed container on a sterile, saline-soaked, nonstick wound dressing pad and transferred to the laboratory for processing, after the bronchoscopy is completed. Gauze or mesh pads are not appropriate for transport because the tissue may become entwined in the mesh material, making extraction difficult and tissue damage likely. As with all small, freshly obtained biopsy specimens, caution must be exerted to avoid prolonged exposure to air because drying artifact can render the specimen uninterpretable.
Figure 3.1 Bronchoscopy. The modern flexible bronchoscope.
For tissue examination by light microscopy, the usual specimen fixation is accomplished using 10% neutral-buffered formalin (4% formaldehyde solution). Biopsies should be submersed in fixative, with an optimal fixative-to-specimen volume ratio of at least 10: 1. For reasons of safety and disposal cost, some laboratories have replaced their formalin solutions with special nonaldehyde fixatives, most of which use alcohol as the primary fixing agent. Of note, all fixatives produce a certain degree of histologic artifact in tissues and cells, and these artifacts can influence the accuracy of the diagnosis. For this reason, it is essential that the pathologist responsible for interpreting the specimen be consulted regarding the type of fixative to be used. Despite some hazards in handling and disposal, 10% neutral-buffered formalin remains the standard agent for lung biopsy fixation.
For transferring bronchoscopic biopsy specimens from carrier or fixative solutions into cassettes for paraffin embedment, a useful device is a polystyrene pipe the with the tip cut off with scissors (Fig. 3.6). This pipetting device allows the operator to transfer delicate specimens without tearing or crushing. Transferring of these specimens using forceps is to be avoided.
When infection is a consideration, bronchoscopic biopsy specimens can be transported directly to the microbiology laboratory for processing.4,19,20 In most scenarios, biopsy samples are sent for histopathologic evaluation and microbiologic studies directly from the bronchoscopy suite or bedside. For endobronchial and transbronchial samples, the optimal number of biopsy specimens varies depending on the radiologic distribution of disease,15,21-23 bronchoscopy findings,8,11,16 and the specific diagnostic entities under consideration.8,24,25 As a general guideline, if the patient is tolerating the procedure well, the greater the number of biopsy specimens, the greater the likelihood of establishing a definitive diagnosis.8,23,25
Figure 3.2 Bronchoscopy. Endoscopic view of right main bronchus with a needle biopsy device inserted (right upper and lower images and left lower image). Note the guide diagram (extreme right), with a red dot indicating the position of the bronchoscope tip.
Figure 3.3 Bronchoscopic biopsy. (A) Cupped biopsy forceps. (B) Bronchoscopic biopsy specimen. The airway epithelium, subepithelial tissue, and muscle wall are typically present with variable cartilage.
Figure 3.4 Bronchoscopic biopsy. Removing the specimen from the device with fine-tipped forceps is not advised. Alternatively, a sterile needle can be used.
Figure 3.5 Lymphangitic carcinoma. Transbronchial biopsy specimen showing lymphangitic carcinoma (arrows) in dilated lymphatic channels included in the sample.
Transbronchial Biopsy
In contrast with endobronchial biopsy, the transbronchial biopsy technique is intended to sample alveolar lung parenchyma beyond the cartilaginous bronchi.8,16,17,26 This technique uses either crocodile-style (Machida) forceps or cupped forceps manipulated by the operator (Fig. 3.7). To obtain the biopsy specimen, the forceps is advanced with the jaws closed into a distal airway until resistance is met. The forceps is retracted slightly and then advanced slightly, with the jaws open. The jaws are then closed, and the forceps is pulled out through the bronchoscope. Advancing the forceps at end-expiration can be helpful in forcing the bronchiolar wall and peribronchiolar lung parenchyma into the mouth of the device. The successful parenchymal biopsy specimen appears finely ragged (Fig. 3.8) and usually measures between 2 and 3 mm in diameter.26-28 Following the transbronchial biopsy, the lung undergoes a repair process that includes fibroplasia similar to that seen in the setting of organizing pneumonia (Fig. 3.9).
As with endobronchial samples, the transbronchial sample is teased from the forceps with a sterile needle, and the same precautions are advised to avoid damage during handling and transfer to fixative or other solution. The truncated pipe The technique also is useful in this setting. Once processed, both types of biopsy specimens should be serially sectioned for thorough microscopic evaluation.2
Several artifacts are encountered in the interpretation of transbronchial biopsies, including crush, bubbles, and sponge effects. Due to the acquisition of the specimen with cupped forceps, nearly all transbronchial forceps biopsies are at risk for crush artifact. It is usually seen at the edge of the specimen and consists of collapse of the alveolar walls (Fig. 3.10A). Bubble artifact occurs when air remains in the tissue while fixing in formalin. Formalin shrinks the tissue, and as it shrinks, the tissue is pressed against the residual air, creating artifactual holes in the tissue specimen, sometimes mistaken for lipoid pneumonia (Fig. 3.10B). Processing transbronchial biopsies in cassettes between sponges generates irregular larger holes in the tissue (Fig. 3.10C and eSlide 3.1). Gentle handling of fresh tissue, agitation in formalin, and avoidance of sponges all can help in generating the optimal specimen for evaluation.
Figure 3.6 Transferring biopsy specimens. For the pipe The transfer method, the pipe The tip is cut off to provide a wider orifice.
Figure 3.7 Transbronchial biopsy. The cupped biopsy forceps with open jaws is commonly used for transbronchial biopsy.
Figure 3.8 Transbronchial biopsy. Low-magnification image of a transbronchial biopsy specimen of generous size.
Figure 3.9 Organizing pneumonia and fibroplasia approximately 1 month after transbronchial biopsy. This patient was profoundly hypoxic and had bilateral mosaic perfusion suggestive of constrictive bronchiolitis. Transbronchial biopsies were negative for interstitial lung disease, and the patient underwent surgical lung biopsy.
Figure 3.10 A variety of artifacts are encountered in the setting of transbronchial biopsies. (A) Due to the use of forceps, nearly all biopsy fragments have the potential to show crush artifact (between arrowheads). (B) Biopsies fixed without any agitation may show bubble artifact that can be confused with lipoid pneumonia. (C) Processing lung biopsy specimens between sponges should be avoided because the sponge can create irregular punched-out spaces in the biopsy with severe associated compression.
Cryobiopsy
Cryobiopsy is a recently developed technique29 to obtain larger portions of lung tissue in an attempt to improve the yield of diagnostic tissue in lieu of an open surgical lung biopsy. Cryobiopsy is performed through the traditional flexible bronchoscope. The cryoprobe is advanced into the area of interest under fluoroscopy to between 1 and 2 cm from the pleural surface. Once in place, the cryoprobe is activated for 3 to 6 seconds. After tissue freezing, the cryoprobe and the bronchoscope are swiftly pulled from the patient. The bronchoscope must also be removed because the cryoprobe with frozen tissue is too large to remove through the working channel of the bronchoscope. Some centers have a second bronchoscopist ready to quickly return to the biopsy site to assess for bleeding. The biopsy is then thawed in saline and then gently placed in formalin (Fig. 3.11A). Each biopsy specimen is often greater than 5 mm, and complete pulmonary lobules are often sampled. This procedure is repeated 3 to 5 more times.
Figure 3.11 Cryobiopsies. (A) Lung tissue from a cryobiopsy procedure after thawing in saline. (B and C) Size comparison of cryobiopsy (B) and traditional transbronchial biopsy (C).
Figure 3.12 Bronchial brush. The conical bronchial bristle brush.
The main histologic benefits of cryobiopsy are larger specimen sizes and lack of crush artifact (Fig. 3.11B and C and eSlide 3.2). Freezing artifact has not been a problem, likely due to the rapid speed of tissue freezing. Several studies have documented the usefulness of cryobiopsy in the setting of diffuse lung disease, with confident diagnoses obtained in more than 75% of cases in some studies.30 the rate of pneumothorax is variable but may be as high as 28%. Other studies have shown no significant difference in adverse outcomes (bleeding, pneumothorax) when comparing cryobiopsy to traditional transbronchial biopsy.31
Bronchial Brushings
Visualized lesions of the airway epithelium can be sampled for cytologic evaluation.8,11,16 the technique involves the use of a conical bristle brush (Fig. 3.12). Under direct visualization, the brush is agitated against the mucosal surface of the airway, forcing cells into the interstices of the bristles (Fig. 3.13). The brush is removed from the bronchoscope and can be applied directly to glass slides. Cells and secretions smeared on slides can be immediately fixed for cytologic evaluation using the Papanicolaou staining method or air-dried for use with the Wright- Giemsa staining technique (this choice is best made in consultation with the pathologist). Immediate fixation of slides is best accomplished by direct immersion in 95% alcohol immediately after smearing of the sample on the slide. Each fixation and staining technique produces characteristic artifacts, and the use of one over the other depends on operator training and preference.
If slides are not available for smear preparations, the brush can be cut off and placed directly into a small vial of sterile saline, which is then shaken vigorously to dislodge cells into the fluid. This fluid sample of suspended cells is sent to the laboratory for Millipore filtration or cytocentrifuge-type application onto slides,8,11,16,32,33 analogous to the handling of washings and lavage specimens, discussed next. With ever-increasing demands for molecular genetic information for use in patient management (typically in the setting of malignant tumor), aliquoting this liquid suspension of cells and saving a portion of it in collaboration with the local pathologist is advisable and may avoid the need for resampling of tumor in cases in which surgical tumor removal is not an option.
Figure 3.13 Bronchial brushing. Application of the brush to the airway mucosa.
Figure 3.14 Bronchial washings. Bronchial washings are obtained by aspiration of sterile saline solution applied near the tip of the bronchoscope. The sample is limited in size and has a variably blood-tinged, frothy appearance.
Bronchial Washings and Bronchoalveolar Lavage
Bronchial washings and bronchoalveolar lavage (BAL) specimens are less lesion-directed sampling techniques and rely on the presence of shed cells within the airways and more peripheral lung.11,34-38 Bronchial washings are obtained by aspiration of sterile saline solution applied near the tip of the bronchoscope (Fig. 3.14). The washings consist of a rather concentrated cellular preparation of bronchial epithelial cells and macrophages with variable amounts of inflammatory cells and mucus. Because of the relatively small sample volume (as with the bronchial brush sample when this is placed into solution), a limited number of assays can be performed on the bronchial washing specimen. Cytologic examination and cultures are the most commonly ordered tests on these samples. If a diagnosis of tumor is likely, saving an aliquot for special studies is prudent. If initial aliquots are judged to be nondiagnostic, the saved sample can be recruited.
Figure 3.15 Bronchoalveolar lavage. A lavage fluid sample with macrophages and neutrophils. (ThinPrep, Papanicolaou stain.)
By contrast, BAL retrieves a large volume of saline that is injected into the airways, allowing more extensive sampling of lung parenchyma and airway luminal secretions. Cell density in the fluid typically is low, and centrifuge or filter techniques are required for microscopic examination.
The lavage procedure is performed by instilling multiple aliquots of sterile saline (20 to 50 mL), followed, after a variable dwell time, by subsequent aspiration of this fluid into a flask or syringe at the bedside. BAL fluid from normal lungs consists primarily of macrophages with a few inflammatory cells37 (Fig. 3.15). A potential advantage of BAL over the bronchial washing technique is the capability to analyze noncellular elements included, such as surfactant content, serum proteins (e.g., albumin, immunoglobulins, enzymes), and mucus.39,40 In current practice, the BAL technique is used primarily in the clinical setting for the diagnosis of infection in the immunocompromised host.39,41-44 However, as a research tool in the study of interstitial lung diseases, BAL has been used extensively for quantitation of cellular components.19,28,39,45-47
In processing the BAL fluid, the operator must determine what types of analysis will be performed in advance. A typical sample might be divided into a number of aliquots, some of which would be sent to the cytopathology laboratory, whereas others would be handled by the general laboratory (microbiology, chemistry, hematology). For cytopathology evaluation, the bronchial washing smears and cytocentrifuge preparations can be air-dried for the Wright-Giemsa staining method. More commonly, in the United States, smears are fixed in an equal volume of either Saccomanno fixative (2% Carbowax and 50% ethyl alcohol) or simply 50% (or greater) ethyl alcohol solution. These fixed smears are then stained using the Papanicolaou method or other staining techniques.
Transbronchial Fine-Needle Aspiration
Transbronchial fine-needle aspiration (TBNA) initially was introduced by Wang and Terry in 198348 as a staging tool in the evaluation of patients with lung cancer. The use of TBNA has expanded considerably since that time49-51 for the diagnosis of both central and peripheral lung lesions, even in the absence of endobronchial abnormalities.51 the specimens generated by this technique are very small, sometimes no more than a drop or two, and when the target is solid tissue, these samples consist of thick cellular material. To prepare direct smear preparations for cytopathologic evaluation and rapid stains for organisms, the needle is removed from the syringe (or other aspiration device) and then reattached after air has been aspirated into the syringe. The air is then rapidly forced out through the needle tip, forcing the sample out of the needle hub and onto a slide. Alternatively, a drop can be expressed directly onto a slide (close to the label end) and smeared using a feathering technique (Fig. 3.16 and eSlide 3.3). The slide smear is then either air-dried or immediately fixed before staining. For microbiology cultures or fluid-based cytocentrifuge preparations, the needle is rinsed directly into culture or cytopathology fixative medium, respectively.
Figure 3.16 Preparing direct cytologic smears from small tissue or fluid samples. The optimal technique for making cytology smears from scrapings or needle aspiration is illustrated. (A) A small amount of sample is placed on the slide near the specimen label end, and a second slide is brought up next to the sample at right angle to the sample slide. (B) After the sample is contacted by the right-angle slide, this "feathering” slide is flattened slightly while pulled forward in a smooth motion toward the operator, as seen in part (C). The feaThere d smear can be fixed immediately or allowed to air-dry, depending on the staining technique chosen. (D) the ideal smear is oval in shape. The specimen shown was stained with the toluidine blue rapid method. (Smear technique courtesy Dr. Matthew Zarka, Mayo Clinic, Scottsdale, Arizona.)
Figure 3.17 Endobronchial ultrasound. A bronchoscope with a distal ultrasound probe and port for needle biopsy.
Endobronchial Ultrasound-Guided Biopsy
In an effort to improve the diagnostic accuracy of TBNA, the endobronchial ultrasound (EBUS) was developed and introduced (Fig. 3.17). EBUS allows real-time ultrasound-guided sampling of lung masses, mediastinal masses, and both mediastinal and hilar lymph nodes for lung cancer staging.52 Optimal diagnostic yield is obtained with three passes of the needle per biopsy site.53 Specimen processing for EBUS is similar to the processing for TBNA. Many institutions use rapid on-site evaluation (ROSE) for cytologic evaluation. A randomized control trial evaluating the need for ROSE concluded that ROSE does not affect the diagnostic yield in EBUS-TBNA but may reduce the number of punctures needed for a diagnosis, as well as the need for other procedures, such as transbronchial lung biopsy.54
Rigid Bronchoscopy
Rigid bronchoscopy is a procedure that has been used for more than 125 years.55,56 With the introduction of the flexible bronchoscope, use of rigid bronchoscopy has declined, but some lesions are more readily biopsied using this device, especially when larger quantities of tissue are required56,57 (Fig. 3.18). The procedure requires use of general anesthesia and expertise in airway management. Large fragments of tumor (or foreign bodies) can be removed, and cautery can be applied to control any bleeding encountered. For large, highly vascular, or mostly necrotic tumors, this may be the most prudent and useful procedure for obtaining diagnostic specimens.
Specimens Obtained by Transthoracic Needle Biopsy and Aspiration
Thoracentesis
Thoracentesis derives its greatest practical application in the evaluation of pleural effusion samples for cells and noncellular elements.58-60 As with BAL fluid examination, a number of specific analyses typically are performed. If collected after hours, the sterile thoracentesis fluid can be stored unfixed at 4°C for processing the next day. The aliquoted thoracentesis fluid specimens are distributed to the appropriate laboratory for analysis (e.g., microbiology, chemistry, hematology). Chemical determinations of glucose, amylase, lactate dehydrogenase, and other analytes are compared with cellular composition determined by cyto- pathology evaluation. The cytocentrifuge or Millipore filter also can be evaluated cytopathologically for the presence of malignant neoplasm. Rapid stains for microorganisms can be performed as indicated.
Figure 3.18 Rigid bronchoscopy. A large obstructing tumor mass is well visualized.
Figure 3.19 Pleural biopsy. (A) the Abrams pleural biopsy needle consists of an outer trocar with a blunt tip and a side cutting port near the tip (right). The trocar is pulled across the parietal pleural edge, hooking this tissue into the side port. (B) the inner cutting cannula is then forced across the cutting port from within, following along a spiral guide path seen on the left end of the outer sheath. (C) the stylet keeps the needle channel closed during initial insertion into the pleural space, there by avoiding the creation of a pneumothorax.
Closed Pleural Biopsy
Available pleural needle biopsy devices include the Cope, Abrams, and Tru-Cut needles that produce a very small biopsy sample (Fig. 3.19). Inflammatory, infectious, and neoplastic diseases of the pleura can be diagnosed using these devices, despite the limitations of biopsy size and somewhat randomness of sampling.61 These small specimens should be handled in a fashion analogous to those obtained from bronchoscopic biopsy techniques (as described previously).
Transthoracic Fine-Needle Core Aspiration and Biopsy of the Lung
In current practice the use of transthoracic needle aspiration biopsy has become commonplace.10,62-67 This procedure typically is performed in the radiology department because biopsy by this method is always performed under radiologic guidance. Samples are similar to those obtained by transbronchial needle aspiration and should be handled accordingly (as discussed previously). Assistance from a cytotechnologist during the procedure is a cost-effective benefit to ensure adequacy of the specimen before termination of the procedure.68 Advances in needle biopsy devices have allowed for better tissue samples and greater likelihood of accurate diagnosis.69-72 If the technique generates a semiliquid sample, this should be handled as described for transbronchial needle aspiration specimens. If a core of tissue is generated (typically 1 mm in diameter), this can be processed like other needle core samples received in surgical pathology (eSlide 3.4). In addition to routine hematoxylin and eosin (H&E)-stained levels for routine evaluation, the histology laboratory also should make a limited number of unstained sections (mounted on slides designed for immunohistochemical stains) at the time of initial sectioning in histology. Having these extra sections available saves time and avoids having to return to the tissue block later when special stains may be necessary, which can be a problem because block resurfacing between sectioning wastes a certain amount of tissue. With the widespread implementation of molecular testing for targeted treatment protocols in lung cancer, tissue preservation has become an important component of in the work-up of lung mass biopsies. Up to 78% of non-small cell carcinoma can be diagnosed on routine H&E alone.73 Some cases need only limited immunohistochemical stains (p40 and TTF-1) to direct further testing. The amount of tumor needed for molecular testing varies on the test being ordered, the methodology being used by the molecular lab, the amount of associated nontumor tissue, the amount of tumor necrosis, and the technical abilities of the molecular laboratory (e.g., if the perform microdissection of tumor). Close communication between the surgical pathologist and the molecular laboratory can decrease the rate of insufficient specimens.
Specimens Obtained by Thoracoscopy
Surgical biopsy of lung parenchyma is indicated in several specific situations:
• A target judged to be too small or in a location with excessive risk to permit biopsy by interventional radiologic techniques74,75
• Peripheral lesions that have eluded endobronchial biopsy attempts1,13
• Suspected interstitial and inflammatory lung disease14,15
The introduction of a high-resolution video endoscopic system has changed the practice of elective thoracic surgery. With this procedure, smaller incisions are made and a thoracoscope with a video camera is introduced along with the instruments (Fig. 3.20). Video-assisted thoracic surgery (VATS) has become the standard of care for obtaining most surgical biopsy specimens. It has been used in the diagnosis and treatment of pulmonary diseases since the early 1990s.76-79 the mortality rate is low, duration of hospital stay is decreased, postprocedure pain is less, and patient recovery is hastened in comparison with standard thoracotomy.77 Specimens measuring 2 to 3 cm across and larger (Fig. 3.21) are easily obtained, and patients often can be discharged from the hospital in less than 24 hours. With VATS, the same thoracic access ports also provide access for sampling ipsilateral lymph nodes that may contain neoplastic disease or other abnormalities.
Although it confers undeniable benefits, VATS usually requires single lung ventilation on the nonoperative side, so all conditions that contradict this maneuver may prevent use of this approach. Tumor deposition and dissemination can be prevented by adherence to well-established oncologic surgical principles.80,81 Meticulous technique is required to avoid laceration of tumor during excision and contamination of the distant pleura, lung, or port incision sites by the instruments. The use of an impermeable Endo Catch bag (Medtronic, Dublin, Ireland) for specimen retrieval is mandatory (Fig. 3.22). A sterile lavage of all port sites is also performed at the conclusion of surgery.
Before any wedge lung biopsy is performed, consultation among the radiologist, chest physician, and thoracic surgeon is essential to ensure appropriate sampling and the identification of ideal locations for biopsy. These considerations are especially important for the investigation of interstitial lung disease, and in patients suspected of having idiopathic pulmonary fibrosis, in which disease tends to localize to the lower lobes. Retrieval of tissue from more than one biopsy site is necessary, preferably from widely separated areas or different lobes. In the ideal scenario, such determinations are based on best surgical judgment combined with the specific characteristics of the disease as identified on clinical and radiologic grounds.
Specimen Processing
Processing of the wedge lung biopsy specimen requires techniques different from those used in handling specimens from other organs. A typical surgical lung biopsy specimen as it is received from surgery is shown in Fig. 3.23. If the wedge tissue sample is to be divided for different types of analysis (e.g., microbiologic cultures, electron microscopy, molecular diagnostic studies), these portions can be separated before routine processing for morphologic assessment. Preparing frozen sections from air-filled lung tissue poses some special problems. Unfixed lung tissue is easily compressed, especially when attempts are made to slice it into thin (typically less than 5 mm) sections unfixed. Severe compression may result in artifactual atelectasis, there by compounding the difficulty of histopathologic assessment (eSlide 3.5). The simplest and most reliable technique for preparing frozen sections is to cut a 5- to 6-mm slab from the biopsy specimen using a fresh scalpel and freezing it without further preparation. For most lung diseases, the frozen sections generated this way are reasonably interpretable. Alternatively, some authors have recommended injecting the actual slab section (not the whole biopsy) with a stabilizing solution before freezing. To accomplish this, a dilute solution of embedding compound can be gently infused into the cut surface of the slab using a 21- to 23-gauge needle attached to a 5-mL syringe.
Figure 3.20 Video-assisted thoracoscopic surgery. (A) Three incisions are made for instruments: video scope, stapler, and manipulator device. The sutured incisions are small (B) and require minimal dressing (C). The drain, seen at upper right in parts (B) and (C), will be removed later.
Figure 3.21 Video-assisted thoracoscopic surgery. (A) Videoscopic view of lung and parietal chest wall, with the lung tissue selected for biopsy (center) held while the stapler isolates this tissue from surrounding lung. The double staple line produced allows safe surgical incision for removal. (B) A closer view of the stapling process.
Figure 3.22 Video-assisted thoracoscopic surgery. The biopsy specimen is transferred into a specimen bag (A) for safe retrieval through the incision (B).
After any intraoperative consultation has been completed, the remainder of the specimen can be prepared for processing using a number of techniques, all designed to restore the normal inflated state of the tissue (VATS specimens are received deflated and stapled closed). We prefer a simple technique that is as good as either of the two more elaborate methods also described here and requires no special equipment or needles: After all staples have been removed from the sample, the specimen is vigorously shaken for 2 minutes in a container half-filled with fixative solution (appropriately sealed with paraffin film) (Fig. 3.24A). This maneuver forces the sample repeatedly against the inner walls of the container and nicely distributes fixative within the elastic
lung parenchyma. In most instances, atelectatic areas are fully restored (see Fig. 3.24B and C). A second technique uses a small volume of carbonated water added to the fixative solution to assist in reexpansion of alveoli (no agitation required). Finally, some authors have proposed using a small-gauge needle and syringe to gently reinflate the sample with fixative. This procedure should be performed only after the staples have been removed from the sample to avoid overinflation. Injection into the cut lung surface is preferable to injecting through the pleural surface (Fig. 3.25A). After fixation of 1 to 2 hours, the specimen can be safely and easily sliced into 3- to 5-mm sections for final processing (see Fig. 3.25B).
Conclusion
Liberal communication among the chest physician, radiologist, pathologist, and thoracic surgeon is strongly advised before embarking on any lung biopsy procedure, especially those associated with procurement of wedge biopsy specimens. Such a multidisciplinary approach is cost effective and increases the likelihood of accurate results.
Figure 3.23 Optimal surgical lung biopsy. The surgical wedge specimen should measure 3 to 5 cm in length and 3 cm in depth (from pleural surface to the stapled edge at specimen midpoint).
Figure 3.24 the simple preferred technique for processing lung biopsy specimens, including both surgical wedge biopsies and transbronchial biopsies. (A) After removing the staple line and sectioning the wedge biopsy, the sections are vigorously shaken in formalin. This infuses the airspaces of the lung with formalin to provide a uniform fixation and restore the lung closer to its anatomic state. (B and C) A comparison of low power side-by-side sections, one which has undergone vigorous shaking (B) and the other which was placed directly in formalin (C).
Figure 3.25 Fixation and sectioning of the surgical wedge biopsy specimen. (A) A tuberculin syringe (with a 23- to 25-gauge needle) can be used for inflating lung wedge biopsy specimens through the cut lung surface after the surgical staples are removed. (B) After the specimen has been shaken in or injected with fixative, submersion in the fixative solution for an additional 1 to 2 hours improves gross section quality and avoids reintroducing atelectatic changes.
Self-assessment questions and cases related to this chapter can be found online at ExpertConsult.com.
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1. Which of the following procedures is/are used in modern pulmonary medicine to obtain tissue specimens from the lungs?
A. Bronchoscopy
B. Video-assisted thoracoscopy
C. Fine-needle aspiration
D. Surgical wedge biopsy
E. All of the above
ANSWER: E
2. Which ONE of the following statements is FALSE?
A. Procurement of clinical and radiologic information is diagnostically more helpful in neoplastic lung disease, compared with nonneoplastic disorders.
B. Specimen size and quality affect the level of diagnostic certainty.
C. Descriptive diagnoses are acceptable in lung pathology.
D. Fine-needle aspiration biopsy of the lung has acceptable specificity and sensitivity compared with diagnosis of pulmonary neoplasms.
E. The marginal quality of any given lung biopsy specimen can be described in the surgical pathology report.
ANSWER: A
3. Which ONE of the following statements is TRUE?
A. Flexible bronchoscopy began in the United States in the late 1980s.
B. Rigid bronchoscopy is no longer performed in Asia.
C. Flexible bronchoscopy requires general anesthesia.
D. Flexible bronchoscopy is best used for examination of the proximal airways.
E. Flexible bronchoscopes have smaller bores than rigid bronchoscopes.
ANSWER: E
4. Modern flexible bronchoscopes:
A. Allow the operator to visualize sixth-order bronchi
B. Are commonly equipped with a cupped forceps
C. May produce biopsies that sometimes include bronchial cartilage
D. None of the above
E. A, B, and C
ANSWER: E
5. Biopsy specimens that are obtained in the bronchoscopy suite:
A. Should ideally be air-dried before submission to the laboratory
B. Can alternatively be placed in fixative solution or transport medium by the bronchoscopist
C. Should be wrapped in sterile dry gauze pads before sending them to the laboratory
D. Are unsuitable for immunohistochemical studies
E. All of the above
ANSWER: B
6. Currently, the standard fixative for lung biopsy specimens is:
A. Ethylene glycol
B. Methacarn
C. 10% formalin
D. Bouin solution
E. A mixture of 20% formalin and 80% ethanol
ANSWER: C
7. Which of the following statements about bronchoscopy specimens is TRUE?
A. They are subject to relatively little artifact due to biopsy technique
B. Air-drying helps to preserve open alveolar spaces in them
C. The optimal number of tissue pieces in them depends on the disease process
D. Fungal cultures cannot be performed using them
E. All of the above
ANSWER: C
8. In transbronchial lung biopsy techniques, which of the following statements is TRUE?
A. Cupped forceps are not used
B. The jaws of the forceps should be open when it is first placed in the airway
C. Biopsies are obtained at end-inspiration of the respiratory cycle
D. The pieces of tissue that are obtained have a smooth cylindrical shape
E. Tissue fragments measure 2 to 3 mm in diameter
ANSWER: E
9. In obtaining specimens for cytopathology, which ONE of the following statements regarding the bronchial brushing technique is TRUE?
A. It uses an instrument resembling a miniature paintbrush
B. Contents of the brush are washed onto glass slides with ethanol
C. Resulting slides cannot be stained with Wright-Giemsa reagents
D. Papanicolaou stain is often applied to the slides
E. Immediate fixation of slides in 10% formalin is recommended
ANSWER: D
10. Molecular characterization of lung tissue can be accomplished using:
A. Bronchial washing specimens
B. Transbronchial biopsy specimens
C. Open lung biopsy specimens
D. All of the above
E. None of the above
ANSWER: D
11. Bronchoalveolar lavage specimens are obtained:
A. After filling the airways of both lungs with sterile saline and waiting 5 minutes
B. Only from adult patients
C. For purposes of tumor diagnosis
D. To evaluate possible lung infections
E. From patients who may have surfactant abnormalities
ANSWER: D
12. In the transbronchial fine-needle aspiration technique of Wang and Terry, the aspirate sample is washed from the biopsy needle with:
A. Air
B. Saline
C. Plasma
D. Ethanol
E. Michel solution
ANSWER: A
13. Which ONE of the following statements regarding rigid bronchoscopy is TRUE?
A. It can be done in an outpatient setting in a physician’s office
B. It is no longer performed in the United States
C. Relatively large foreign bodies can be extracted with it
D. Necrotic lung tumors should not be accessed with it
E. It was introduced as a new method in the year 1925 and abandoned in 1995
ANSWER: C
14. Which of the following statements regarding thoracenteses is TRUE?
A. They are performed for relief of symptoms in cases of pleural effusion
B. They yield specimens that can be kept unspoiled at 4°C for several hours
C. They can be used for chemical and enzymatic analyses
D. They are suitably performed in cases of suspected intrapleural tumor
E. All of the above ANSWER: E
15. Why is it advisable for histotechnologists to prepare four to six unstained glass slides of small tissue specimens?
A. They can be used later for biochemical analysis of the tissue
B. The medicolegal risk attending these specimens mandates that all of them should be sent out for extramural consultation
C. The tissue can be scraped off the slides to reconstruct the lesion they contain in three dimensions
D. All of the above
E. None of the above
ANSWER: E
16. In the clinical procedure abbreviated as VATS, what does the “V” stand for?
A. Virtual
B. Vacuum
C. Video
D. Vesalius
E. Vivisection
ANSWER: C
17. Which of the following statements regarding open wedge biopsies of the lungs is/are TRUE?
A. Intercollegial consultation is strongly recommended
B. They are performed primarily for the treatment of peripheral lung cancers that measure greater than 5 cm in diameter
C. One biopsy specimen from one lung is acceptable for diagnosis
D. They are inferior to transbronchial biopsies for diagnosis of interstitial lung diseases
E. They can now be done without general anesthesia ANSWER: A
18. Which of the following methods can be performed very successfully using paraffin blocks of lung tissue?
A. Electron microscopy
B. Flow cytometry
C. Molecular cytogenetic studies
D. Microbiologic cultures
E. All of the above
ANSWER: C
19. What is the recommended method for performing frozen section microtomy on fresh lung tissue?
A. Freezing a 5- to 6-mm thick slab of tissue cut with a fresh scalpel
B. Embedding the fresh tissue in agar
C. Infusing the specimen with Bouin solution using a needle and syringe
D. Slow-freezing the specimen with a drop in temperature of no more than 5°C per minute
E. Filling the airways with latex before cutting the sections
ANSWER: A
20. Which of the following techniques can be used to optimize fixation and histologic visualization of atelectatic lung tissue?
A. Shaking the specimen in a sealed container that is half full of fixative
B. Removing all surgical staples before fixation and prosection
C. Adding a small volume of carbonated water to the fixative solution
D. Insufflating the tissue with fixative using a needle and syringe, after removing surgical staples
E. All of the above
ANSWER: E
Case 1
Transbronchial biopsy with sponge artifact (eSlide 3.1)
a. History—A 39-year-old female without previous medical history presents with acute shortness of breath over the past 2 days. During her evaluation in the emergency room, her respiratory status declines and she ultimately requires intubation. Imaging studies show bilateral infiltrates. Bronchoscopy with biopsy is performed.
b. Pathologic findings—Despite this being a generous amount of lung tissue for evaluation on transbronchial biopsy, it is quite difficult to interpret. The tissue is extensively crushed, and the airspaces are not clearly defined. Close inspection of the tissue reveals numerous irregular punched-out spaces with surrounding compressed lung tissue. These are evidence of compression between sponges during fixation. In the background There is evidence of acute lung injury and a marked increase in the number of eosinophils.
c. Diagnosis—Acute eosinophilic pneumonia with marked laboratory- induced histologic artifact suggesting use of sponges during fixation.
d. Discussion— The pulmonologist’s use of the biopsy forceps during the transbronchial biopsy procedure introduces crush artifact into every biopsy taken. The laboratory should not only avoid processing methods that induce artifacts but can also attempt to minimize the artifacts from the procedure. Transbronchial biopsy specimens should be handled gently and should not be compressed by sponges, tissue bags, or other constrictive processing methods. Instead, gentle agitation in formalin shortly after the biopsy is obtained can help to reexpand the crushed tissue from the forceps. These two steps can dramatically improve the histology, ensuring the best possible material on which to make a diagnosis.
Case 2
Cryobiopsy with diagnosable interstitial lung disease
(eSlide 3.2)
a. History—A 62-year-old male presents with chronic shortness of breath. Computed tomography imaging shows extensive subpleural reticulation with a basal predominance. No air trapping is identified. Using strict criteria, the imaging is read as a “possible” radiographic usual interstitial pneumonia pattern. A cryobiopsy is performed. Three cryobiopsy samples are taken and placed into different cassettes. A representative slide is scanned for review.
b. Pathologic findings— The biopsy is quite generous, measuring more than 5 mm. From low magnification There is advanced geographic fibrosis in some areas and perfectly normal lung in others. Scattered throughout the interface between the fibrosis and normal lung are several fibroblast foci. No granulomas or lymphoid hyperplasia is identified.
c. Diagnosis—Usual interstitial pneumonia pattern on a cryobiopsy.
d. Discussion—Cryobiopsy is increasingly used for the diagnosis of interstitial lung disease in lieu of surgical lung biopsy. In many cases the pathology is sufficient to provide a working diagnosis and institute treatment, thus saving the patient significant morbidity associated with surgical lung biopsy. The major complication rate for cryobiopsy appears to be similar to the rate for traditional transbronchial biopsy.
Case 3
Endobronchial ultrasound-guided fine-needle aspiration with adenocarcinoma (eSlide 3.3)
a. History—A 74-year-old male presents with an incidentally found speculated left upper lobe mass and mediastinal lymphadenopathy. Endobronchial ultrasound-guided fine-needle aspiration is performed on the 4R lymph node.
b. Pathologic findings— The cytologic preparation is quite cellular with a robust background of lymphocytes, confirming a lymph node sampling. In addition to the lymphocytes, there are numerous cohesive aggregates of epithelial cells. These cells show an increased nuclear to cytoplasmic ratio, prominent nucleoli, and irregular nuclear boarders.
c. Diagnosis—Metastatic adenocarcinoma involving the 4R lymph node.
d. Discussion—Ultrasound guidance has improved the diagnostic yield on endobronchial fine-needle aspiration procedures because it allows direct visualization of the needle in the target by the endoscopist. These procedures are increasingly being used not only for diagnosis but also for the procurement of tumor for molecular analysis in attempt to identify a targetable mutation.
Case 4
Needle biopsy with mucinous adenocarcinoma (eSlide 3.4)
a. History—A 54-year-old female with no past medical history is found to have a 3.2-cm circumscribed area of “ground glass” in the peripheral right lower lobe. A transthoracic needle core biopsy is performed.
b. Pathologic findings—Numerous cores of tissue are available for evaluation. There is a proliferation of well-differentiated cells spreading
along the alveolar walls in a lepidic fashion. In some areas, there is architectural distortion of the background lung architecture. The cells have mildly enlarged basally located nuclei with abundant cytoplasmic mucin production.
c. Diagnosis—Invasive well differentiated mucinous adenocarcinoma.
d. Discussion—These tumors are deceptively well differentiated based on their cytologic features. The challenge with mucinous adenocarcinoma is that it tends to spread throughout the airways and can present at a high stage despite the low-grade cytology. Unless There is a history of prior malignancy, it is not recommended to obtain immunohistochemical stains on mucinous adenocarcinoma of the lung. They are often TTF-1 negative and CDX-2 positive, which only complicates the diagnosis and uses up tissue that might be needed for molecular studies. Mucinous adenocarcinomas are most likely to be KRAS mutated but may also show ROS-1 mutations.
Case 5
Poorly processed video-assisted thoracic surgery biopsy with crush and atelectasis (eSlide 3.5)
a. History—A 33-year-old female presents with a recent hospitalization for shortness ofbreath requiring oxygenation. She has a 24-pack year smoking history but quit 3 months ago. Imaging studies show extensive upper lobe scarring. A surgical wedge biopsy is performed.
b. Pathologic findings—Sections show relatively preserved pulmonary architecture. From low power There is significant compression of one end of the biopsy, creating compressive atelectasis. Some air bubbles are seen fixed into the compressed tissue. In the background, at higher power, one can appreciate that much of the airspace filling disease is in the form of lightly pigmented macrophages. In addition, at higher power, one can appreciate relatively diffuse areas of dense collagenous fibrosis within the interstitium. The airways show marked small airway remodeling with small airway dropout, marked mucostasis, and bronchiolectasia. A few nodular foci of inflammation are present. Some of the inflammatory cells include eosinophils, lymphocytes, and plasma cells. There are numerous histiocytes, some of which are reminiscent of Langerhans cells. A significant component of organizing pneumonia is not appreciated.
c. Diagnosis—Advanced smoking-related changes, including smoking- related interstitial fibrosis, a desquamative interstitial pneumonia-like reaction, active pulmonary Langerhans cell histiocytosis, and chronic small airway remodeling. Compressive atelectasis secondary to fixation of the wedge biopsy in formalin without removal of the staple line and inflation with formalin.
d. Discussion—A variety of surgical- and pathology-related artifacts could be encountered in surgical lung biopsies. The pathology laboratory can take steps to minimize the artifacts. All surgical lung biopsies are significantly crushed/compressed during the stapling procedure. Prompt removal of the staple line is the most effective step to take because this will release the tissue prior to formalin fixation. If possible, the specimen can be submerged in formalin and also shaken/ agitated for a minute. This helps to infuse the spongelike lung tissue with formalin and remove the air. This helps to return the lung to its physiologic state and lessen the crush and bubble artifacts that can make the interpretation challenging.