Cytology Primer for Endosonographers

Key Points

  • Communication between the endosonographer and the cytopathologist is the key to a successful endoscopic ultrasonography-guided fine-needle aspiration (EUS FNA) service.

  • A cytopathology service should be involved early in the planning process for establishing an EUS FNA service.

  • Using an algorithmic approach to diagnosing a patient will facilitate a correct diagnosis.

Conceptual breakthroughs, based on developed theories, and discoveries in science bring accolades. Advances in the biotechnology field are signs of the dominance of creative imagination expressed through technology over abstract conceptual thinking. Despite such subtle differences in the concepts put forth, most clinicians involved in patient care agree that advances in the biomedical sciences have significantly broadened horizons and have redefined patient management.

The field of endoscopic ultrasonography (EUS)-guided fine-needle aspiration (FNA) should be viewed as no different. It could be said that the keystone events in the development of modern endosonography were the conceptualization and production of flexible endoscopes for human use in the late 1950s. In the 1980s, ultrasound probes were attached to endoscopes, and Doppler imaging capability was introduced. These improvements allowed better visualization of lesions and an understanding of vascular flow. These powerful scopes could characterize lesions not only of the luminal gastrointestinal tract, but also of the gastrointestinal tract wall, the peri-luminal lymph nodes (intrathoracic and intraabdominal), the pancreas, the liver (mostly the left side), the left kidney, the spleen, and the adrenal glands. The list continues to grow. EUS imaging alone, however, may not be sufficient to differentiate neoplastic from nonneoplastic and a benign from malignant lesion. Further advances in technology made since the early 1990s permit the performance of FNA under EUS guidance. The ability to obtain cytologic material safely under real-time visualization makes this a powerful modality that offers an opportunity for prompt and accurate diagnosis and staging.

The outcome of the EUS FNA diagnosis depends on effective collaboration between the cytopathologist and endoscopist. The best results are achieved by those clinicians who really believe in cytology for their own patients and who work in close cooperation with cytologists. Thus an understanding of relevant issues by both endosonographers and cytopathologists involved in obtaining and interpreting cytologic specimens optimizes the diagnostic yield. When such visions are synchronized, the diagnostic performance of EUS FNA far exceeds expectations. As predicted earlier, this technique has now become a standard of care at many institutions and continues to replace other modalities for tissue diagnoses, staging, and adequate management of patients.

The objective of this chapter is to help both endosonographers and cytopathologists learn the technical aspects of cytology procedures and to understand the basic principles of interpretive cytopathology diagnosis. Thus the chapter reviews pertinent technical aspects that may influence cytology interpretation and affect outcome. It also discusses the algorithmic approach and salient cytologic features of benign and malignant lesions commonly sampled by EUS FNA.

Technical Aspects of Endoscopic Ultrasonography That Improve Diagnostic Yield

Fundamental to the success of EUS FNA is the procurement of adequate cells to provide the most effective diagnosis and potential for additional management. This requires careful planning and understanding of factors that can affect cellularity of the target lesion.

Preliminary Planning

Ideally, an interested pathologist should be involved in the development of the EUS FNA service from the earliest stages of the planning process. This includes such crucial factors as the location of the endosonography suite, the type of instrument and needle used, the personnel involved, the scheduling of FNA, the type of preparation, the transport medium, the need for immediate cytologic evaluation (ICE) or rapid onsite specimen evaluation (ROSE) for determination of adequacy and diagnosis, the need for performing ancillary studies, and the role of the procedure in the patient management algorithm ( Table 22.1 ). Further planning should also involve ordering of supplies, stocking and provision of the FNA cart or cabinet, or maintenance of a permanent small space for supplies in the endoscopy suite area.

TABLE 22.1

Factors to Consider in Preliminary Planning for Cytology Services

Factor Details
Type of biopsy Needle core or cytology (fine needle)
Size of needle 25 G, 22 G, 19 G, or other
Fixation or processing for cores Formalin, other
Type of preparation of cells for FNA Direct smears, transport media (proprietary, culture media [RPMI-1640], formalin, other)
Type of smear Air-dried, alcohol-fixed, or both
Personnel GI suite staff, laboratory staff, training
Immediate cytologic assessment Cytopathologist, cytotechnologist, advanced trainee, not performed
Database archives for cytology information Diagnosis, number of passes, pathologist, type of smears prepared, cell block available, special studies

FNA, Fine-needle aspiration; GI, gastrointestinal.

The type of tissue specimen preparation (direct smear, liquid-based cytologic preparation, cell block, core biopsy, or a combination) depends on institutional practice, staffing issues, and the physical distance between the pathologist and the endoscopy suite, in addition to the relative sensitivity, specificity, and diagnostic accuracy of the various choices. If needed, based on physical location and personnel availability, one should strongly consider use of real-time telepathology system for ICE or ROSE. Developing adequate skills for accurate interpretation of EUS samples not only depends on an experienced cytopathologist but may also require additional specific experience in interpreting these samples in a high volume service. Experience indicates that cytopathologists who are specifically interested in gastrointestinal diseases tend to be more effective in providing accurate diagnoses.

For the pathologist and laboratory staff, a comprehensive understanding of their direct role in the EUS procedure and the patient care algorithm ensures appropriate support. Diagnostic strategies depend on whether the procedure is a screening test, a diagnostic test in a patient who may not undergo further diagnostic workup, or a test to procure material for performance of ancillary studies to enhance patient management decisions.

A further preliminary planning step is consideration of database archives of cytology and diagnostic data. In combination with the EUS characteristics of lesions and other clinical information, these data can provide valuable feedback regarding diagnostic accuracy, individual practitioner competency, utility of ICE, and other quality assurance measures.

Professional staff should be properly trained and should understand the limitations of their expertise and of the technique. In the United States, both the technical and the interpretive services in the cytology laboratory are regulated at state and federal levels by the provisions of the Clinical Laboratory Improvement Amendments, the Laboratory Accreditation Program of the College of American Pathologists (CAP), and others. Such mandatory and voluntary standards ensure high-quality laboratories.

The following sections discuss technical factors that may improve diagnostic yield for EUS biopsy procedures, including needle type and size, suction or “capillary” aspiration, number of passes, and direction of passes. These factors are listed in Table 22.2 .

TABLE 22.2

Technical Aspects That May Positively Influence Diagnostic Yield

Technical Feature Advantage Disadvantage
Preliminary planning Optimal laboratory support None
Endoscopist skill More likely to procure adequate specimen None
Pathologist skill Few if any false-positive or “atypical” diagnoses None
Core biopsy Histologic diagnosis Possible more tissue injury
Tissue for special stains No capacity for on-site evaluation for adequacy
Does not require on-site laboratory personnel for specimen processing or evaluation
Aspiration biopsy More cells Few disadvantages
Risk of inadequate sample for some lesions or sites
Smaller needle size Less tissue injury Relatively fewer cells
Suction Retrieves more cells Increases bleeding in tissue
May compromise some cell features
More passes More cells Injury to tissue
Cytopathologist in room Specimens adequate for diagnosis Time and cost
Air-dried and alcohol-fixed smears Complementary stains yield optimal nuclear and cytologic detail Increased technical effort required
Cell block Tissue available for special stains Not a stand-alone preparation; best in combination with smears

Fine-Needle Aspirates

Fine-needle aspirates are used widely for EUS, computed tomography (CT), and other image-guided biopsy techniques, as well as for percutaneous biopsies of palpable masses. The material contained within a fine-needle aspirate is usually smeared onto slides, and the resulting monolayer of cells is immediately fixed or dried and stained. Material obtained from a fine needle is generally dispersed as single and small groups of cells, rather than intact tissue cores. As the preparation is not sectioned, the cells represented on an aspirate smear are intact. Thus cells round up or splay out, depending on how they are treated in further processing steps. The monolayer smears prepared from fine-needle aspirates are very important to decipher details of the nucleus and cytoplasm that is superior to many other modalities.

Recently new developments have allowed acquisition of core biopsies utilizing endoscopy ultrasound-guided route. These samples have the advantage of providing tissue architecture.

Choice of Needles

Fine needles are defined as needles that have 22 G or smaller bore. Varying sizes of EUS instruments and needles are available on the market, and the choice of needle may also influence cytology findings. The cutting edge of the needle plays a role in obtaining samples; for example, a beveled edge requires less force in comparison with circular edges. Similarly, needle sizes also have an impact on the procurement of tissue samples. In current practice, EUS needles range from 19 G to 25 G. Contrary to intuitive thinking that larger size is always better for FNA samples, sometimes the smaller-bore needle provides better sampling.

Several prospective studies and meta-analysis of published literature have been attempted to determine the impact on cell yield and diagnostic performance of samples obtained by the various EUS FNA needle types. Some of these investigators suggested that needle aspirates from 25-G needles provide less hypocellular or acellular and bloody specimens, have better diagnostic performance, and perhaps induce less tissue injury in comparison with samples from a 22-G needle. Other investigators, however, could not independently confirm this finding and found either no or minimal difference between 22-G and 25-G needles with regard to the ability to render a diagnosis.

FNA samples are also increasingly used for ancillary studies. In a study designed to determine the optimum needle size and number of passes to obtain material for RNA quantitation, the number of cells obtained from needles of varying sizes was counted. With 10 needle excursions into a tumor, 32,000 cells were obtained with a 25-G needle. Although large numbers of cells are important for some tests, such as RNA extraction, it is generally accepted that diagnoses can be made on smears containing fewer than 100 cells. Investigators have suggested that a larger needle (e.g., 22 G) may be useful for lesions associated with less risk of complication or that require large numbers of cells for classification. Unraveling of underlying molecular targets that may affect diagnosis, prognosis, or therapeutic predictions are increasingly being characterized. This has resulted in increased utilization of small tissue samples in performance of molecular tests such as fluorescence in situ hybridization (FISH) analysis, pyrosequencing, and newer platforms to generate genetic profiles utilizing techniques such as next-generation sequencing (NGS). As a unique technique, the authors have also destained Diff-Quik–stained smears and performed FISH analysis to detect specific chromosomal translocations in lymphomas to improve diagnostic performance. This method has the clear advantage of making a morphologic determination and then using the same cells for detection of characteristic chromosomal translocations to clinch the diagnosis.

Given the tradeoff between more cells and more complications with larger fine needles, the choice of needle size should be based on the site and type of lesion to be aspirated. Indications for a smaller needle (e.g., 25 G) include patients with coagulopathy, organs in which leakage of fluid or air may occur, organs in which tissue trauma may increase complications (e.g., pancreas), and vascular organs or lesions. A smaller needle size decreases potential complications such as bleeding into the tissue and hemodilution or obscuring of the cytology sample by excessive blood. Smaller needles also cause less tissue damage and thus possibly less risk of pancreatitis.

Needle Core Biopsy Versus Fine-Needle Aspiration

Unlike many cytopathologists, for several reasons some clinicians and surgical pathologists believe that a tissue core yields unequivocally better diagnostic material. This belief perhaps stems from the concept that tissue cores will result in adequate samples with fewer needle passes, they do not involve on-site specimen assessment, they provide architecture, and ancillary studies can be performed on these samples. It is also true that needle core biopsies (14 G to 19 G) have been used for a long time to obtain tissue samples. Sections made from these core biopsies are thin, 3- to 5-μm slices of the tissue that, when stained and viewed microscopically, show cells or portions of cells within their intact tissue stroma. Most histopathologists are very familiar with this method of tissue-based assessment and therefore promote the use of core needle biopsy samples.

Conversely, however, one should also be aware that analysis of tissue core may not always provide adequate diagnostic clues. Tru-Cut biopsy also induces greater tissue injury than a fine-needle biopsy and is considered more invasive. Such considerations should deter clinicians from using large-bore Tru-Cut needles routinely. It is also true that needle core biopsies can pose greater challenges for diagnosing well-differentiated carcinomas of the pancreas in comparison with FNA samples. In preliminary analyses, the success of FNA sampling using EUS guidance led to a sharp decline in performance of percutaneous needle core biopsies and CT-guided FNA. Such a change dramatically altered practice management decisions for pancreatic neoplasms.

A possible explanation for the failure of core needles to sample the lesion may be the attributes of the lesion itself, given that a larger needle may deflect from the surface of a firm or rubbery lesion. In addition, a Tru-Cut biopsy represents a single pass into the tissue and is not able to sample the lesion widely without further passes into the tissue. The use of larger needles increases the risk of bleeding and complications, although these risks remain very low. In addition, technical limitations of the currently available EUS-guided Tru-Cut biopsy equipment limit the anatomic regions that can be sampled for biopsy successfully.

The use of EUS-guided Tru-Cut biopsies has recently demonstrated a significant promise in review of lymphadenopathy as well as from other solid organs. Tru-Cut biopsy is useful not only to establish the diagnosis of lymphoma but also to characterize cellular architecture, which is more important in disorders such as follicular center cell lymphomas. Tru-Cut biopsy also may be more useful in cases in which flow cytometry results can provide false-negative results, such as large B-cell lymphomas. EUS-guided Tru-Cut biopsy may also be helpful in establishing the difficult diagnosis of Hodgkin lymphoma, in which morphology is varied and often challenging to identify. Needle core biopsies are also very useful for lesions rich in stroma such as gastrointestinal tract stromal tumors. Epithelial rich lesions such as most carcinomas are amenable to aspirations.

Recently, newer biopsy needles have been introduced to procure samples with minimal fragmentation, thereby retaining architecture. Histologically, an ideal core has uninterrupted structure or architecture, whereby cell groups maintain their interface with extracellular matrix or stroma. Architecture can be crucial to the definitive diagnosis and/or staging. Three needles are being utilized more frequently in today’s practice which include reverse bevel needles (Procore, Cook Endoscopy, Winston Salem, North Carolina), fork-tip needles (Shark Core, Medtronic Corp., Boston, Massachusetts), and Franseen tip needles (Acquire, Boston Scientific, Marlborough, Massachusetts). Initial assessment of Procore needles; 25-G, 22-G, 19-G fork-tip needles (Shark Core, Medtronic Corp., Boston, Massachusetts); and the 22-G Franseen tip needle (Acquire, Boston Scientific, Marlborough, Massachusetts) has demonstrated a strong promise in tissue acquisition studies. Their performance from pathologist review perspectives, however, has not been adequately assessed.

The decision to obtain cores instead of, or in addition to, aspirates rests on certain factors, including the available equipment and personnel, the training and expertise of the pathologists and staff, and the endoscopist’s preference. Each type of biopsy has advantages and disadvantages that must be considered for individual lesions or patients. Overall, FNA is considered a more sensitive diagnostic method, and it can be complemented by core biopsy or cell block.

To Apply or Not to Apply Suction

For many fine-needle biopsies, suction is applied to the needle to attempt to increase cell yield. This is the origin of the term fine-needle aspirate, which is often used more generally for any fine-needle biopsy. The purpose of suction is not to draw cells into the needle, but rather to “hole” the tissue against the cutting edge of the needle. Suction should be turned off before the needle is withdrawn.

In another technique, the cells are obtained without applying suction. The lumen is filled with cells by the direct cutting action of the needle through the tissue or capillary action. A study of 670 superficial and deep lesions sampled by biopsy with a fine needle without suction showed that diagnostic material was obtained in more than 90% of the cases. Specific to EUS FNA, a study by Wallace and colleagues found no difference in suction versus no suction in terms of overall diagnostic yield for lymph nodes, but these investigators noted excess blood in the specimens to which suction was applied. Another study demonstrated that EUS-guided fine-needle sampling with suction increased the number of slides (17.8 ± 7.1 slides) needed to be prepared, as compared with a significantly ( P < .0001) reduced number of slides to be prepared for samples in which no suction was applied.

In general, applying suction to the needle increases cellular yield but potentially increases artifact and blood, especially in vascular organs and lesions. Suction is commonly used because the increased cellular yield of specimens often outweighs the disadvantages. Some clinicians attempt up to three passes without suction and add further passes with suction if the cellular yield is low. Although no suction improves analysis, the choice to use or not to use suction should be dictated by the type of lesion.

When a large amount of blood is aspirated and the specimen clots, a less desirable but useful salvage of material is to gently microdissect the clot or fragment the clot with a scalpel blade or needle tip. The fragments are then lifted from the slide and are placed in formalin for subsequent cell block preparation. Forceful smearing of the clot to disperse the cells may cause significant crush artifact and may render the cells uninterruptible.

Number of Passes

A pass usually comprises 10 or more needle excursions or movements of the needle to-and-fro once the needle is within the lesion. The number of passes needed to obtain diagnostic material depends on multiple factors, including experience of the endosonographer, location of the lesion, type of lesion, cellularity of the lesion, and risk of complications. Many investigators suggest that after a certain number of passes, the procedure reaches a state of diminishing returns for obtaining diagnostic cellularity.

The landmark study was performed earlier where the issues of number of passes and specimen adequacy rates were more carefully investigated in 204 cases. In our early analysis of more than 204 cases, diagnostic cellularity was obtained after five passes in more than 90% of cases. It also emerged in this study that the rate of diminishing returns was reached earlier for lymph nodes and later for the pancreas. For solid pancreatic lesions, adequate cellularity was achieved with fewer numbers of passes when the lesion was smaller (≤25 mm) compared with larger lesions. This study also demonstrated that after five passes, lymph nodes offered little benefit in obtaining diagnostic cells. It was also evident that for lymph nodes, a mean of only three passes was needed for obtaining diagnostic cellularity. LeBlanc and colleagues determined that at least seven passes were needed in pancreatic lesions to obtain a sensitivity and specificity of 83% and 100%, respectively, although only five passes were needed in lymph node aspirates for a respective sensitivity and specificity of 77% and 100%. In recent years, better technique and experience and fellowships in advanced endoscopies have led to a reduction in the number of passes needed to procure adequate diagnostic cellularity.

A well-known advantage of real-time image-guided biopsies, especially EUS, is the ability to direct the needle to a small point of interest. Selection of the exact site of biopsy may influence the cytologic yield. Biopsy of the necrotic center of a tumor may be nondiagnostic, whereas the edge may contain viable tumor cells. Conversely, biopsy of the edge of a pancreatic carcinoma may show only chronic pancreatitis, a common reactive change in the surrounding pancreatic tissue.

Depending on the anatomic site, directing the needle to specific portions of the lesion may be advantageous. Metastatic tumor in lymph nodes may be histologically more apparent in the subcapsular sinus, but in lymph node aspirates evaluated by EUS FNA, aspiration of the edge of the node did not increase the likelihood of a correct diagnosis. Nonetheless, because EUS allows visualization of the lesion, biopsy of a necrotic area can be avoided, and as discussed later, on-site evaluation of the specimen can provide guidance to another location if the first site is necrotic.

A main advantage of the FNA technique is the wide sampling of a lesion by maneuvering the needle in different directions with each back-and-forth movement. Small redirections of the needle to make a fan shape will result in sampling of new areas of the lesion each time. Utilization of the fanning technique, as described in Chapter 20 , in comparison to the standard multiple pass technique to obtain cells helps reduce the number of passes and improves diagnostic cellularity rates. Repeated needle excursions in the same direction, along the same needle tract, result in biopsy of the blood or fluid that can fill the area with blood.

Immediate Cytologic Evaluation

One way to ensure adequate material from an FNA procedure is the use of ICE ( Video 22.1 ). The goal of ICE is to provide real-time feedback about the content and quality of the smears, to reduce the number of nondiagnostic or atypical biopsies, and to maximize the efficiency of the procedure. We, as well as other investigators, have demonstrated that ICE yields increased highly reliable preliminary diagnosis and will also help triage the specimen for performing ancillary studies. Other investigators have demonstrated that specimen adequacy is more than 90% when a cytopathologist is present in the endoscopy suite for ICE. Such high specimen adequacy rates drop when cytopathologists are not present in the endoscopy suite for ICE. In a direct comparison of EUS FNA procedures performed by the same endoscopist at two institutions, with and without a pathologist present during the procedure, ICE was more likely to result in a definitive diagnosis and less likely to involve an inadequate specimen. Most false-negative EUS results are caused by inadequate sampling, which may necessitate a second procedure. It is also true that the most effective way to reduce a sampling error is ICE.

Video 22.1

Video of On-Site Processing of Specimen Procured by Endoscopic Ultrasonography-Guided Fine-Needle Aspiration

A retrospective analysis was conducted of changes noted after the transition from CT-guided FNA to EUS-guided FNA sampling from the pancreas. Cytopathologists were present to provide ICE in an endoscopy suite, whereas this practice was not in place for samples obtained under CT guidance. The results demonstrated that EUS FNA provided more definitive diagnoses and fewer unsatisfactory or equivocal diagnoses. The investigators were also able to procure additional samples for ancillary studies. Such efforts at other institutions have also seen greater than 90% specimen adequacy rates and reductions in equivocal diagnosis. When ICE is performed, selected air-dried slides are stained in the endoscopy suite or an adjacent room and are reviewed immediately by the pathologist, so that feedback can be given to the endoscopist regarding the adequacy of the pass. If diagnostic material is present, additional passes are not made, and the procedure is stopped. If the smears are nondiagnostic, further passes are made. If there are no cells or only necrosis, the needle can be redirected for the next pass, and the procedure can be continued until adequate material for diagnosis is obtained.

In addition to minimizing the number of passes needed to obtain diagnostic material, another advantage of ICE is the triage of specimens for special studies. Such a practice may allow for the procurement of samples for ancillary studies such as lymphoma workup or for cell block when the initial smears show a tumor that may need classification by immunohistochemistry, in situ hybridization, or other studies for better patient management. Thus obtaining additional directed passes is encouraged for creating an adequate cell block.

Although ICE clearly improves diagnostic yield, this practice is variable throughout the world. The use of ICE is influenced by the physical location of the laboratory and gastrointestinal suite, personnel, and cost issues. Reluctance of a pathologist to attend EUS FNA procedures may relate to lack of time and inadequate reimbursement for the time investment required.

Unfortunately, however, lack of will on the part of consumers of these services has not helped change the conventional stance on reimbursements in the United States. Instead, the reimbursement rates are increasingly becoming more cost prohibitive. All institutions and regions of any country are different, and they need to develop their own cost-effective strategies for the sake of providing optimal health care for their patients.

In an attempt to minimize the impact of the lack of ICE, different investigators, with variable success, have investigated alternatives. These alternatives include assessment of cellularity by visual inspection, performance of smears and evaluation by endosonography personnel, and the use of services of advanced cytotechnologists following adequate training or advanced trainees in cytopathology. In this context, the use of dynamic telecytology for adequacy assessment was also investigated.

Regardless of whether ICE is used or not, an adequate sample is the foundation of the diagnosis. The needle must be placed into the lesion; the technical aspects of the sampling must be optimized to obtain cells for evaluation; and the smears must be free of crush, drying, staining, or other artifact, and obscuring blood, inflammation, or necrosis.

Factors Associated With Improved Cytologic Preparation

The material from EUS-guided biopsy can be prepared in many different ways, each of which has advantages and disadvantages. Some preparations are complementary, and two or three types are often prepared from the same biopsy specimen. The following sections define preparation of air-dried and alcohol-fixed smears, cell block, and the stains used for highlighting various cell features.

Cytology Smears and Cell Block

A smear slide is the standard method of preparing cells obtained from a fine-needle biopsy for viewing. As in a blood smear, the biopsy material is dispersed or “smeared” onto a glass slide, stained, and viewed as individual cells. For EUS FNA, after the needle is removed from the endoscope, the tip is placed near the frosted end of a labeled slide, and a single small drop is expressed onto the slide by slowly advancing the stylet into the needle. Dropping the material from a distance, squirting, or spraying it onto the slide can result in drying of the specimen and unwanted artifact. A second slide is then drawn over the drop of material, to pull the material into a monolayer. The technique requires practice. If the smear is too thick, the cells are obscured by one another or by background cells; if too much pressure is applied, the cells are artificially disrupted from their normal microarchitecture or are lysed. Imperfect smears may reduce the diagnostic yield. Another technique is called the “butterfly technique,” where a drop is placed in the center of the slide and two slides are placed at right angles such that the material spreads between two slides by capillary action. One slide is then air-dried and the counterpart is immediately fixed in alcohol for additional stains. No pressure is applied. This technique requires less technical expertise without compromising the quality of diagnosis.

Should one acquire tissue core biopsies, then one should firmly touch the core tissue to transfer cells on the slides. Firmly applying pressure to the core may result in squashing the tissue which may or may not provide optimal information.

In contrast to smears, a cell block is a preparation in which the cells are placed into a liquid medium or fixative, transported to the laboratory, spun into a pellet, formalin fixed, paraffin embedded, and selected for standard hematoxylin and eosin (H&E) staining. This routine formalin fixation and paraffin embedding is not optimal for preserving cytologic detail. A cell block is often made from leftover material rinsed from the needle. Its value as an adjunct to diagnosis can improve if an additional directed pass is obtained at the end of the procedure. This technique is highly recommended, especially for lesions that may require special stains.

Air-Dried or Alcohol-Fixed Smears

Generally, smears prepared from FNA material are either air-dried or alcohol-fixed. Air-dried smears are stained rapidly (using a modified Romanowsky stain [e.g., Diff-Quik]) and are typically used for ICE. Some institutions use H&E or rapid Papanicolaou (Pap) stains for ICE.

Diff-Quik–stained, air-dried smear preparations highlight intracytoplasmic material and extracellular substances. Alcohol fixation causes cells to shrink and round up, but it preserves nuclear features and is followed by Pap or H&E staining. The Pap stain highlights nuclear detail and chromatin quality, in addition to demonstrating the keratinization of squamous cells. The cytoplasm appears more transparent in Pap-stained slides. Slides can be fixed in preparation for a Pap stain by immersing or spraying them with alcohol. The Pap and Diff-Quik stains are complementary, and optimal cytologic detail is provided when both alcohol-fixed and air-dried smears are prepared from the FNA.

Transport Media and Liquid-Based Preparations

Samples are frequently collected in transport media for subsequent preparations. Although many media are available, Hank’s balanced salt solution is preferred. This medium allows for the preparation of cytospins and cell blocks, and should one require lymphoma consideration later, this medium can also be used for flow cytometric analysis. For consideration of any lymphoma workup, many institutions also collect their samples in RPMI 1640. This is also a useful medium to collect for cytogenetic analysis, as well as gene rearrangement studies.

Liquid-based cytology is now firmly embedded in clinical practices. Oftentimes when ICE/ROSE is not possible, these samples can be placed in the transport medium for cytologic interpretation. Several studies have suggested that this can obviate the need for ICE/ROSE. It is also becoming more evident that for pancreatic cysts utilizing this approach is more beneficial, which is increasingly being investigated. Currently two methods have been approved by the Food and Drug Administration: ThinPrep (Cytyc Co, Marlborough, Massachusetts) and SurePath (TriPath Inc., Burlington, North Carolina). There are slight differences between the two methods but both offer advantages of monolayer cell dispersion, elimination of obscuring mucus and blood, and consistent cell preparation without artifacts of preparation, as noted with smear preparations.

These techniques, however, increase the cost of preparation and cannot be used for ICE. Because the preparations may disaggregate cells (loss of architecture) and alter some cytologic details, they offer challenges to interpretation. Some of the proprietary liquid fixatives contain methanol, a coagulative fixative (rather than a protein cross-linking fixative such as formalin), which may lead to suboptimal fixation for immunohistochemistry. Liquid-based cytology preparations do not fare as well as direct smear preparations. However, liquid-based cytology offers a viable alternative when ICE is not a consideration. Samples from pancreas prepared using liquid-based cytology preparations demonstrated smaller cell clusters, smaller cell size in comparison with air-dried smears, better nuclear characteristics, and diminished or absent mucin. Furthermore, these samples could not be used at a later time for flow cytometry analysis. Such considerations should be taken into account during selection of transport media and preparations. A detailed model of an optimized EUS FNA procedure is shown in Table 22.3 .

TABLE 22.3

Optimized Endoscopic Ultrasonography Fine-Needle Aspiration Model Technique

Stage Description
Preparation When the procedure is scheduled, arrangements are made for the cytology technician and pathologist to be at the site. Clinical findings are discussed with the pathologist at the start of the procedure. The locations of the lesions or other details must be known, based on previous imaging studies. Conscious sedation is provided to the patient with intravenous meperidine and midazolam.
Needle preparation The stylet is removed completely from a 22-G EUS FNA needle, and the needle is flushed with heparin. Air is then flushed through the needle to expel the excess heparin. The stylet is replaced, and the needle is ready for use. The needle may also be straightened manually between passes if necessary.
Radial EUS A radial echoendoscope is first used for an overview of appropriate anatomic landmarks. The location of lesions is noted.
Linear array EUS FNA The radial echoendoscope is replaced with a linear array echoendoscope. The scope is advanced to the distance at which the lesion of interest was identified with radial endosonography. The lesion is visualized, and color Doppler is used if there is concern about intervening blood vessels. The EUS FNA needle is inserted and fastened to the biopsy channel of the echoendoscope, and then it is advanced just slightly beyond the scope into the gut lumen. At this point, the stylet is retracted approximately 1 cm. The needle is passed into the lesion. The stylet is replaced into the needle to expel any tissue from normal structures and then is removed completely, and a suction syringe is attached. Sampling is performed with and without suction. The needle is moved into various locations throughout the lesion (“fanning the lesion”) to improve sampling. After approximately 20 back-and-forth movements, the suction is turned off, the needle is retracted back into the catheter, and the entire assembly is removed.
Expressing material on slide A dedicated cytology technician holds the end of the catheter over a labeled glass slide. The needle is advanced approximately 1 cm from the catheter by the endoscopy technician, and a stylet is slowly advanced back into the needle. This produces a controlled passage of drops of material out from the tip. The cytology technician alternately places drops onto a slide and into transport medium. Finally, the needle is flushed with a few milliliters of saline and then air to expel any remaining material into the liquid medium.
Preparing and staining cytologic material Slides are prepared depending on the amount of material. As rapidly as possible, the drops of aspirated material are spread downward onto the slides by using another clean glass slide. Half of the slides are air-dried, and the remaining slides are immediately immersed in 95% ethyl alcohol for later Papanicolaou staining. The air-dried slides are stained with Diff-Quik stain for immediate cytologic evaluation by the pathologist (see later in the chapter). When the procedure is finished, an additional dedicated pass may be placed in transport medium (e.g., Hank’s balanced salt solution) and transported to the laboratory, and a cell block is prepared. The material in cell suspension is centrifuged into a pellet, to which thrombin is added. The pellet is resuspended, and the resulting clot is removed, wrapped in lens paper, placed in a tissue cassette, fixed in formalin, and routinely processed for paraffin embedding and H&E or immunostaining. If indicated, material for flow cytometric immunophenotyping or other studies is removed from the medium, and the cell block is prepared. The alcohol-fixed slides are stained with a standard Papanicolaou stain.
Immediate cytologic evaluation A pathologist, advanced trainee, or experienced cytotechnologist examines air-dried Diff-Quik–stained slides prepared at the site and provides assessment of specimen adequacy. Based on this report, the endoscopist may continue with the same technique or may change needle position to procure more tissue. Immediate cytologic evaluation also helps triage the specimen or obtain additional passes for special studies.

EUS, Endoscopic ultrasonography; FNA, fine-needle aspiration; H&E, hematoxylin and eosin.

Cytology Interpretation

Evaluation of the biopsy begins the moment material is expressed from the needle onto a slide or into a fixative. An adequate aspirate, or one that is likely to yield a diagnosis, is cellular, so that when placed on the slides and smeared out, a finely granular quality is apparent. In contrast, in a hypocellular or purely bloody smear, the thin sheen of material is smooth. When the material is placed in fixative, visible particulate matter or cloudiness is usually present. Mucus, pus, and necrosis may also be grossly apparent.


Once under the microscope, the smear is first assessed for adequacy. For an aspirate to be interpretable, it must be free of technical artifacts and must contain cells for evaluation. A global assessment of cellularity as a measure of adequacy, however, may be misleading in FNA, because the number of cells relates to the lesion. For example, aspiration of neuroendocrine tumors usually yields highly cellular smears, whereas aspiration of a gastrointestinal stromal tumor (GIST) may yield few cells but both may be equally adequate for diagnosis.

For diagnostic nongynecologic cytology specimens, a sample is adequate when it explains the clinical situation or target lesion. The aspirator must be certain that the lesion has been sampled, and the pathologist must be able to interpret the slides. The concept of the “triple test” is also applicable to EUS FNA. The clinical, imaging, and FNA findings should agree and correlate on whether the lesion is benign or malignant. Some lesions have characteristic morphologic features, and therefore a cell number criterion for such tumors is not a requirement.

Diagnostic Evaluation of the Slide

Whether on-site or in the laboratory, the cytotechnologist or pathologist begins the slide evaluation by assessing the cell types, cell arrangement, and cellular features on the smear. Central to a cytology diagnosis is the appearance of the nuclear and cytoplasmic features of individual cells; these are quite distinct, depending on the lesion sampled. No single feature is diagnostic of malignancy, but rather the composite picture of cell type, microarchitecture, and nuclear and cytoplasmic characteristics determines the diagnosis. It is useful to know the common pathologic diagnoses as well as the characteristic of the normal tissue in the region sampled ( Table 22.4 and Fig. 22.1 ).

TABLE 22.4

Some Common Endoscopic Ultrasonography Cytologic Diagnoses in Specific Sites

Site Cytologic Diagnoses
Lung Adenocarcinoma, squamous carcinoma, small cell carcinoma, granuloma or infection
Esophagus Squamous carcinoma, adenocarcinoma, granular cell tumors, leiomyoma or other spindle cell tumors (GIST or neurofibroma)
Stomach Carcinoma, carcinoid, GIST, MALT lymphoma
Pancreas Ductal adenocarcinoma, chronic pancreatitis, autoimmune pancreatitis, pancreatic endocrine neoplasm, metastatic carcinoma, intraductal papillary mucinous neoplasm, mucinous cystic neoplasm, solid pseudopapillary tumor
Rectum and perirectal lymph nodes Metastatic adenocarcinoma or squamous carcinoma, GIST
Liver Metastatic carcinoma, melanoma, sarcoma, lymphoma, primary hepatocellular tumors

GIST, Gastrointestinal stromal tumor; MALT, mucosa-associated lymphoid tissue.

Fig. 22.1

Common lesions of the mediastinum. MPNST, Malignant peripheral nerve sheath tumor.

As in histologic sections, order and aesthetics reign in cytologic preparations of benign tissue. The appearance and composition of a benign aspirate reflect the various cell populations in normal tissue. Epithelial cells are round to oval, have moderate to abundant cytoplasm, and are cohesive. Benign epithelial cells show evidence of differentiation. Squamous cells acquire keratin as they mature, whereas their nuclei become progressively smaller and darker (pyknotic). A benign superficial squamous cell exfoliated from the esophagus has a large, polyhedral shape, with a small, uniformly dark, nucleus described as an “ink dot” ( Fig. 22.2 ). The cytoplasm is orange-pink to blue, depending on the degree of keratin accumulation. Benign, mature squamous cells appear single, unless they are from the deeper layers of the epithelium, in which case they may remain together as large sheets of cells with less keratinization of the cytoplasm. Benign glandular epithelium from the stomach ( Fig. 22.3 ), intestine, and pancreas also demonstrates an orderly arrangement of differentiated cells with organ-specific variations. In smears, duodenal epithelium consists of folded or draped sheets of columnar cells, with interspersed goblet cells appearing as clear spaces among the absorptive cells ( Fig. 22.4 ). Glandular cells are polarized, with the nucleus present at one end of each cell in the sheet of epithelium. The cytoplasm may be filled with a single mucin droplet (the goblet cell), smaller more finely divided vacuoles, or other secretory products such as zymogen granules. Classically, benign columnar epithelium has a honeycomb pattern. Changing the microscope plane to focus reveals the hexagonal borders of the apical cytoplasm and polarized, orderly nuclei at the base of the honeycomb sheet. In contrast, benign stromal or mesenchymal cells have elongated nuclei and usually abundant cytoplasm. Occasionally, small vascular structures are visible in smears of benign tissue.

Feb 19, 2020 | Posted by in GASTROENTEROLOGY | Comments Off on Cytology Primer for Endosonographers

Full access? Get Clinical Tree

Get Clinical Tree app for offline access