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 bronchi^17,18 (Fig. 2-2). Biopsy of visualized mucosal lesions most commonly is performed using cupped forceps (Fig. 2-3A) 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. 2-3B). 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. 2-4) 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. 2-5).

Figure 2-2 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 2-3 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 2-4 Bronchoscopic biopsy. Removing the specimen from the device with fine-tipped forceps is not advised. Alternatively, a sterile needle can be used.

Figure 2-5 Lymphangitic carcinoma. Transbronchial biopsy specimen showing lymphangitic carcinoma (arrows) in dilated lymphatic channels included in the sample.

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 reexpand any crush artifact induced by the biopsy procedure, and if done in a carrying medium, the supernatant can be aliquoted and sent for cytopathogic 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.

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 non-aldehyde 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 pipette with the tip cut off with scissors (Fig. 2-6). This pipetting device allows the operator to transfer delicate specimens without tearing or crushing. Transferring of these specimens using forceps is to be avoided.

Figure 2-6 Transferring biopsy specimens. For the pipette transfer method, the pipette tip is cut off to provide a wider orifice.

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

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. 2-7). 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. 2-8) and usually measures between 2 and 3 mm in diameter.^26–28

Figure 2-7 Transbronchial biopsy. The cupped biopsy forceps with open jaws is commonly used for transbronchial biopsy.

Figure 2-8 Transbronchial biopsy. Low-magnification image of a transbronchial biopsy specimen of generous size.

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 pipette technique also is useful in this setting. Once processed, both types of biopsy specimens should be serially sectioned for thorough microscopic evaluation.²

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. 2-9). Under direct visualization, the brush is agitated against the mucosal surface of the airway, forcing cells into the interstices of the bristles (Fig. 2-10). 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.

Figure 2-9 Bronchial brush. The conical bronchial bristle brush.

Figure 2-10 Bronchial brushing. Application of the brush to the airway mucosa.

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,^{8,11,16,29,30} 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.

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.^11,31–35 Bronchial washings are obtained by aspiration of sterile saline solution applied near the tip of the bronchoscope (Fig. 2-11). 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 2-11 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.

BAL, by contrast, 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 (20–50 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 cells³⁴ (Fig. 2-12). 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.^36,37 In current practice, the BAL technique is used primarily in the clinical setting for the diagnosis of infection in the immunocompromised host.^36,38–41 As a research tool in the study of interstitial lung diseases, however, BAL has been used extensively for quantitation of cellular components.^{19,28,36,42–44}

Figure 2-12 Bronchoalveolar lavage. A lavage fluid sample with macrophages and neutrophils. (ThinPrep, Papanicolaou stain.)

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’s 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 1983⁴⁵ as a staging tool in the evaluation of patients with lung cancer. The use of TBNA has expanded considerably since that time^46–48 for the diagnosis of both central and peripheral lung lesions, even in the absence of endobronchial abnormalities.⁴⁸ Endoscopic ultrasound imaging has increased the accuracy of TBNA in recent years and is becoming a standard part of the TBNA procedure in many settings.^49–51 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. 2-13). 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 2-13 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, Once 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 feathered 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 of Dr. Matthew Zarka, Mayo Clinic, Scottsdale, AZ.)

Rigid Bronchoscopy

Rigid bronchoscopy is a procedure that has been used for more than 125 years.^52,53 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 required^53,54 (Fig. 2-14). 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.

Figure 2-14 Rigid bronchoscopy. A large obstructing tumor mass is well visualized.

Thoracentesis

Thoracentesis derives its greatest practical application in the evaluation of pleural effusion samples for cells and noncellular elements.^55–57 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 cytopathology 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.

Closed Pleural Biopsy

Available pleural needle biopsy devices include the Cope, Abrams, and Tru-Cut needles that produce a very small biopsy sample (Fig. 2-15). 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.⁵⁸ These small specimens should be handled in a fashion analogous to those obtained from bronchoscopic biopsy techniques (as described previously).

Figure 2-15 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 thereby avoiding the creation of a pneumothorax.

Transthoracic Fine-Needle Core Aspiration and Biopsy of the Lung

In current practice, the use of transthoracic needle aspiration biopsy has become commonplace.^10,59–64 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.⁶⁵ Recent advances in needle biopsy devices have allowed for better tissue samples and greater likelihood of accurate diagnosis.^66–69 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. In addition to routine hematoxylin and eosin–stained sections and any number of sectioning levels that typically are obtained for routine evaluation, the histology laboratory also should make four to six 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.

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 Figure 2-19. If the wedge tissue sample is to be divided for different types of analysis (e.g., microbiological 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, thereby compounding the difficulty of histopathologic assessment. 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 2-19 Optimal surgical lung biopsy. The surgical wedge specimen should measure 3–5 cm in length and 3 cm in depth (from pleural surface to the stapled edge at specimen midpoint).

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). 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. 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. 2-20A). 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. 2-20B).

Figure 2-20 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, Once the specimen has been shaken in or injected with fixative, submersion in the fixative solution for an additional 1–2 hours improves gross section quality and avoids reintroducing atelectactic changes.