chapter 12 Reporting Terminology and Accuracy Reactive Hyperplasia (Without Specific Etiology) Benign Lymphadenopathies with Specific Etiology Inflammatory/Infectious Conditions with Characteristic Fine-Needle Aspiration Findings B-Cell Lymphoma, Unclassifiable, with Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma Peripheral T-Cell Lymphoma, Unspecified Anaplastic Large Cell Lymphoma Adult T-Cell Leukemia/Lymphoma Post-Transplant Lymphoproliferative Disorders Enlarged lymph nodes are a prime target for fine-needle aspiration (FNA). In an adult, lymph nodes greater than 1 to 2 cm are an immediate source of concern and, unless the cause is evident, the enlarged node should be aspirated. Although FNA is readily applicable to children also,1 lymphadenopathy in children and young adults is common and usually due to reactive hyperplasia; for this reason, it is often watched and not aspirated unless the node is very large or persistent. FNA is particularly useful in patients with deep-seated lymphadenopathy (e.g., mediastinal, retroperitoneal, abdominal), for which surgical intervention carries the risk of significant morbidity. Even with superficial (e.g., cervical) lymphadenopathy, using FNA avoids uncommon but serious morbidity associated with lymph node excision, like accessory spinal nerve injury.2 FNA has been successfully used for the primary diagnosis of lymphoma in many institutions.3–5 In some individuals FNA is ideal for this indication, particularly those with rapidly progressive disease, an oncologic emergency (e.g., spinal cord compression, airway compromise, superior vena cava syndrome), deep or surgically inaccessible nodes, advanced age, and/or comorbid clinical conditions that preclude surgical biopsy or excision.6 Conversely, when excisional biopsy is less problematic, a larger amount of tissue might be desired at academic institutions for correlative research studies involving emerging technologies like proteomics and genomics, although FNA may sometimes be able to obtain material for these studies as well.7 • important architectural and/or vascular patterns are lost in some entities: • progressive transformation of germinal centers (PTGC) • Human immunodeficiency virus lymphadenopathy (HIVAL) • nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) • T-cell–rich large B-cell lymphoma (TCRLBL) • angioimmunoblastic T-cell lymphoma • diffuse large B-cell lymphoma (DLBL) arising in follicular lymphoma The principle guiding the cytopathologist is the same as that guiding the surgical pathologist: the integration of clinical information, light microscopic analysis, and results of ancillary studies into a final diagnosis. The full value of FNA is only achieved with this integrated approach.8 The cytopathologist who performs the FNA, in fact, has a great opportunity (and responsibility) to incorporate clinical history and physical findings into the final diagnosis9 and to judiciously set aside material for ancillary studies when indicated. Indeed, although FNAs are performed by pathologists and clinicians, pathologists have an advantage in that they can perform an on-site adequacy assessment and repeat the procedure until adequate material is obtained for both light microscopy and ancillary studies, which, for suspected lymphoma cases, may include flow cytometry, cell block (for immunocytochemistry), fluorescence in situ hybridization (FISH), karyotypic analysis, and molecular genetic studies. If there is evidence of an infection (neutrophils, granulomas, necrosis, or visible organisms), a portion of the specimen can be submitted for microbiologic culture. The major relative contraindication is a severe coagulation disorder; however, in patients with mild coagulopathy, pressure can be applied to a superficial node after aspiration to prevent a hematoma, and appropriate blood products can be given in the case of a deep-seated node aspiration. Complications of lymph node FNA are rare. The most common is a hematoma. Data are sparse on the frequency of a hematoma, but experience suggests that it occurs in less than 1% of cases. Tissue artifacts attributable to FNA (focal hemorrhage with organization; segmental or total infarction) are seen in only 4% of excised lymph nodes10 and rarely preclude histologic analysis.11 A pneumothorax can result from aspiration of a deep axillary, low cervical, or supraclavicular lymph node if the pleural space is entered inadvertently. Terminology for reporting lymph node aspiration results is similar to that used for most cytologic specimens. Results are classified into several broad categories: non-diagnostic (unsatisfactory), negative, atypical, suspicious, or positive for malignant cells. The frequency of non-diagnostic (unsatisfactory) results ranges from 5% to 15% of cases12–15 and is site and size dependent. For example, it is more difficult to obtain adequate material from a small, deep axillary node than a large cervical lymph node. FNA of lymph nodes has high sensitivity and specificity in the distinction between a benign and malignant lesion. Accuracy estimates for lymph node FNA vary because of local variations in technique and referral patterns, but most investigators report over 90% accuracy in the diagnosis of metastatic tumor to lymph nodes, and a positive predictive value of almost 100%.15 Some oncologists question the utility of FNA for the primary diagnosis of lymphoma,16 even though most studies performed since flow cytometry became readily available show a sensitivity for recognizing non-Hodgkin lymphoma that is higher than 80%,3–5,8,17,18 with specificity greater than 90%. The importance of using modern practice guidelines when evaluating an FNA for the possibility of lymphoma can not be overemphasized. At a minimum, the cytologist must have a working knowledge of the 2008 and any subsequent World Health Organization (WHO) classification;19 some of the sample must be allocated for immunophenotyping (IP); and a close interaction with hematopathologists must be fostered. Under such circumstances, the accuracy of FNA for the diagnosis of non-Hodgkin lymphoma is excellent. Accuracy is even higher when FNA is performed for recurrent, as opposed to newly diagnosed, non-Hodgkin lymphoma. Regarding subclassification, some lymphomas are easier to subtype precisely than others. IP either by flow cytometry20,21 (preferred) or immunocytochemistry plays a vital role in the diagnosis and subtyping of lymphomas; without it, the accuracy of FNA is severely diminished. Lymphomas that have characteristic immunophenotypes, like small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), and lymphoblastic lymphoma, are easier to recognize than those that do not.17 Differentiating the so-called “small cell lymphomas” from each other and from reactive hyperplasia by morphology alone is not recommended.12–14 In addition, FISH and molecular techniques (discussed below) are playing an increasingly important role as adjuncts to FNA, particularly in the diagnosis and subtyping of non-Hodgkin lymphoma. Clonality in B-cell lymphomas is usually documented by demonstrating light chain restriction: a clonal expansion of B cells that preferentially express either κ or λ immunoglobulin light chains. Clonal B-cell proliferations identified by flow cytometry almost never occur in reactive lymph nodes, but exceptions have been documented.22 Therefore, it cannot be overemphasized that all aspects of the case—morphology, immunophenotype, and clinical features—must be taken into consideration before issuing a diagnosis of lymphoma. Immunophenotyping to determine B-cell clonality and document antigen expression can usually be performed by flow cytometry,13,17,21,23–25 although immunocytochemistry on cell block preparations can also be used.26,27 Although flow cytometry is preferred, each method has its advantages (Table 12.1). TABLE 12.1 COMPARISON OF FLOW CYTOMETRY AND IMMUNOCYTOCHEMISTRY FOR THE ANALYSIS OF LYMPHOID MARKERS To fully characterize a lymphoma, between 12 and 15 antibodies may be needed,28 but often fewer are sufficient. If the sample is sparsely cellular, the cytologist can assist the flow cytometry or immunocytochemistry laboratory by using cell morphology to select an appropriate, more limited panel of antibodies. The routine application of flow cytometry has markedly improved the diagnostic accuracy of FNA.29,30 In a flow cytometer, a liquid suspension of single cells is made to flow in single file through a laser beam. As each cell traverses the beam, it scatters light in different directions. With immunocytochemistry, the presence of cell markers is probed on cells attached to a glass slide. We prefer to perfom immunohistochemistry on formalin-fixed, paraffin-embedded sections from cell block preparations (because this is technically similar to the more familiar procedure for tissue specimens), but some antigens, notably κ and λ immunoglobulin light chains, are difficult to evaluate in fixed specimens. For this reason, unfixed (air-dried) cytocentrifuge preparations can be attempted on lymph node FNAs when lymphoma is a consideration, but, even in such preparations, background staining can be high. Commonly, slides are prepared by cytocentrifugation onto a 6-mm circle on a glass slide. The limited area to be evaluated conserves antibodies and facilitates interpretation of results and is particularly appropriate when the specimen is too scant for cell block preparation. Some of the most commonly used markers are listed in Table 12.2. Immunostaining methods vary slightly from one laboratory to another.26,27 TABLE 12.2 USEFUL CORE IMMUNOCYTOCHEMICAL MARKERS FOR THE DIAGNOSIS OF LYMPHOPROLIFERATIVE DISORDERS The polymerase chain reaction (PCR) amplifies tiny amounts of a defined region of DNA, provided that sequences surrounding the region of interest are known. The technique requires repeated cycles of denaturation and DNA synthesis using oligonucleotide primers, a heat-resistant DNA polymerase, and the four nucleotides. The oligonucleotide primers are synthesized to complement DNA sequences flanking the region of interest. Typically, 20 to 60 cycles of denaturation and synthesis over several hours result in a tremendous amplification of a homogeneous set of the DNA sequence of interest. PCR is fast (1 to 2 days), does not require radioactive probes, and can be performed on formalin-fixed cell blocks. Owing to its extraordinary sensitivity, however, it is prone to false-positive results; rigorous controls and critical review of the results are vital. PCR is a useful test for immunoglobulin and T-cell receptor rearrangements as markers of clonality.25,31–35 PCR for clonality is redundant for B-cell neoplasms that are shown to be monoclonal by immunophenotyping, but it is useful in select circumstances: It can be used as a confirmatory test when flow cytometry suggests a T-cell neoplasm, when cells fail to survive flow cytometry processing, or when unfixed cells are unavailable for flow cytometry.31,36 With FISH, DNA probes for specific chromosomal regions, labeled with a fluorochrome such as rhodamine, are hybridized with intact nuclei. Smears, cytocentrifuge preparations, and, especially, thinlayer slides are ideal substrates, because the intact cells on cytologic preparations allow optimal signal detection. DNA probes are particularly useful for demonstrating translocations,37 deletions, and gene amplifications (e.g., trisomies). Most practitioners recommend that cells be allocated for PCR and FISH, in case they are needed, in addition to flow cytometry, in every case of suspected lymphoma.38 Gene expression profiling using microarray analysis may find a role in the cytologic diagnosis of lymphoma. Aspirates of suspected non-Hodgkin lymphoma can be rinsed in a ribonucleic acid (RNA)–stabilization reagent, and gene expression profiling performed by hybridizing RNA to gene chips.39 Among other potential applications, gene expression profiles can distinguish among different prognostic subtypes of diffuse large B-cell lymphoma (DLBL).40 A dispersed, isolated cell pattern is typical of lymphoid cells, but there are exceptions. Smear thickness, clotting, and suboptimal spreading technique may cause pseudo-clustering of lymphocytes. Aggregates of lymphocytes adherent to follicular dendritic cells occur in follicular hyperplasia and follicular lymphoma (FL),41,42 where they are known as dendritic-lymphocytic aggregates or intact follicles. Conversely, some nonlymphoid tumors (e.g., melanoma) lack cohesion and mimic malignant lymphoma. Detached fragments of lymphoid cell cytoplasm, visible in most smears of lymphoid (both benign and malignant) tissue, are given the fanciful (and imprecise) name lymphoglandular bodies.43 In Romanowsky-stained smears they are a pale blue or blue-gray, and may contain tiny vacuoles; they are much more difficult to see in Papanicolaou-stained smears. Rarely, similar findings are seen in a small number of nonlymphoid lesions such as small cell neuroendocrine carcinoma,44 but, in general, the absence of lymphoglandular bodies makes it unlikely that the cells are of lymphoid origin. There is great variety in the cell types encountered (Fig.12.1). Small lymphocytes with round nuclei and coarsely textured chromatin usually predominate, but they are admixed with other cells. Centrocytes are intermediate-sized lymphocytes with irregular or cleaved nuclei, inconspicuous nucleoli, and scant cytoplasm. Centroblasts are large cells with round, vesicular nuclei, one to three peripheral nucleoli, and a narrow rim of basophilic cytoplasm. Immunoblasts are large cells with fine, open chromatin; one very prominent, centrally located nucleolus; and moderate to abundant, pale to basophilic cytoplasm. Some have a perinuclear clear zone. Tingible-body macrophages are very large phagocytic cells with a spherical or ovoid nucleus, finely granular chromatin, a small distinct nucleolus, and voluminous debris-laden cytoplasm. Dendritic cells are also large, with pale oval nuclei, small nucleoli, and cytoplasmic processes; they are usually binucleated or multinucleated and may appear epithelioid if the cytoplasmic processes are not prominent. Figure 12.1 Reactive lymphoid hyperplasia. Dendritic-lymphocytic aggregates (also known as follicular center fragments or intact follicles when tingible-body macrophages and capillaries are present), are clusters of dendritic cells, small lymphocytes, centrocytes, and centroblasts41 (Figs. 12.2, 12.3). Distinguishing between fragments that do or do not contain tingible-body macrophages and capillaries does not appear to have diagnostic value. These aggregates break the rule that lymphoid lesions lack cell cohesion; they may be seen when follicular hyperplasia is a prominent component of the lymphoid proliferation and must not be confused with a cohesive metastatic neoplasm. Figure 12.2 Reactive lymphoid hyperplasia. Figure 12.3 Reactive lymphoid hyperplasia. Because little stroma exists to retain lymphoid cells during aspiration, large numbers of cells are obtained, resulting in highly cellular smears. Thus, high cellularity in a lymph node FNA does not correlate with malignancy. In most examples of reactive lymphoid hyperplasia, small lymphocytes vastly outnumber centroblasts and centrocytes, which are themselves more numerous than immunoblasts, plasmacytoid lymphocytes, and tingible-body macrophages. The major challenge is to exclude a lymphoma or one of the benign lymphadenopathies with specific etiology. Knowledge of the clinical background is critical; an FNA diagnosis of reactive hyperplasia should be avoided in the elderly45 and in patients with markedly enlarged (e.g., greater than 3 cm) or deep-seated nodes and/or multiple enlarged nodes. Because architectural pattern is not available, cell size, smear composition, and, (most importantly) immunophenotype are used as discriminators. An often cited, helpful morphologic feature is the heterogeneity of the cell pattern: In non-Hodgkin lymphoma the range of lymphoid forms and other cell types is sometimes more narrow. In practice, however, there are many exceptions. Hodgkin lymphoma, follicular lymphoma (FL), marginal zone lymphoma (MZL), post-transplant lymphoproliferative disorders (PTLDs), some T-cell lymphomas, and T-cell-rich large B-cell lymphoma (TCRLBL) have a heterogeneous appearance that mimics reactive lymphoid hyperplasia. The distinction in such cases relies on identifying rare atypical forms (e.g., Reed-Sternberg cells or lymphocytic and histiocytic [L & H] cells in Hodgkin lymphoma) or the immunophenotype, which is useful whenever non-Hodgkin lymphoma is a consideration (clinically or cytologically). In the special case of the patient who has received a solid organ or bone marrow tranplant, in situ hybridization for Epstein-Barr virus (EBV)–encoded RNA (EBER) can be helpful in identifying a PTLD.46 Castleman disease (CD) is a rare form of lymph node hyperplasia and is divided into hyaline-vascular type (usually unicentric and asymptomatic) and plasma cell type (usually multicentric and often associated with constitutional symptoms, elevated interleukin-6, and human immunodeficiency virus [HIV] and human herpesvirus 8 [HHV8] infection). The most common sites of involvement in the unicentric type are the mediastinum and lung, but peripheral lymphadenopathy is the rule for patients presenting with the multicentric type. In the hyaline-vascular type, tissue sections reveal small, hyalinized germinal centers and broad expansion of the mantle zone. Large, pleomorphic follicular dendritic cells are seen. In the plasma cell type, the interfollicular areas contain sheets of mature plasma cells. Although these findings are characteristic, they are not specific, and ultimately CD is a diagnosis of exclusion. There are few descriptions of the cytomorphology of CD in FNA specimens, but some investigators suggest that dysplastic follicular dendritic cells may be a clue to the diagnosis.47,48 The presence of tissue fragments containing branching capillaries and/or hyaline material has been proposed as a cytologic hallmark, but similar vascular branching can be seen in reactive lymphoid hyperplasia. Many believe that a specific diagnosis by FNA is not possible even with immunophenotypic analysis.47 Fortunately, the clinical significance of this difficulty is minimal: In the unicentric type, a nonspecific FNA diagnosis in a symptomatic patient would eventuate in excision of the node, curing the disease, whereas in the multicentric type, blood tests for HHV8 and/or interleukin-6 (or an HHV8 immunostain of an FNA cell block) could provide the diagnosis (in the appropriate clinical context). A caveat, however, for the FNA cytologist is that HHV8 preferentially infects immunoglobulin M (IgM) λ-expressing B cells, so that the resulting virally driven proliferation could appear clonal by immunophenotyping—simulating lymphoma—but is in reality polyclonal,49 as could be demonstrated by molecular (PCR) testing for immunoglobulin gene rearrangement. Lymphadenopathy caused by Toxoplasma gondii is most commonly cervical and usually self-limited, but it can mimic lymphoma clinically. The histologic changes are highly suggestive of this entity: follicular hyperplasia with small aggregates of epithelioid histiocytes that hug the periphery of germinal centers, and zones of monocytoid B-cell hyperplasia. These architectural features are largely unappreciated in smears, which resemble reactive hyperplasia. The diagnosis is usually confirmed by serologic titers. It is only when organisms are seen—a rare occurrence—that a confident cytologic diagnosis can be issued.50,51 The organisms occur as bradyzoites within enlarged finely granular histiocytes, or as free tachyzoites lying within an exudate, and their presence can be confirmed by immunocytochemistry51 or PCR. Progressive transformation of germinal centers (PTGC) is a condition marked by follicle expansion, disruption, and/or replacement by mantle cells, which are small- to intermediate-sized lymphocytes with round or irregular nuclei, inconspicuous nucleoli, and scant cytoplasm. As an architectural disruption, PTGC is impossible to identify by FNA. By flow cytometry, one-third of PTGC cases show a significant proportion of CD4+CD8+ (“double positive”) T-cells, which can be a clue to the diagnosis, but such populations can also be seen in T-cell lymphoma, thymoma, and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL).52 In fact, PTGC is often seen in patients with NLPHL, but most patients with PTGC do not develop lymphoma. Sarcoidosis is a systemic granulomatous disease of unknown cause affecting young to middle aged adults that is more prevalent in African Americans. A wide variety of tissues may be involved, but the lung and lymph nodes of the mediastinum are most often affected. Peripheral lymphadenopathy is most common in the head and neck.53 Noncaseating granulomas are a characteristic but nonspecific feature of sarcoidosis—they are also encountered in fungal, bacterial, and mycobacterial infections; hypersensitivity conditions; neoplasms such as Hodgkin lymphoma and T-cell lymphoma; the drainage pathway of a malignancy; and exposures to foreign material—and the diagnosis (whether by surgical pathology or FNA) is always one of exclusion and clinical correlation. An important practical point is that granulomas are often sparse in FNAs in the late phase of sarcoidosis (due to fibrosis); such aspirates are usually very hypocellular. Indeed, sarcoidosis is one of the commonest explanations for the paradoxical finding of a very hypocellular or acellular aspirate from a large node (provided a targeting error has been excluded). The key component of a granuloma is the epithelioid histiocyte, with its elongated, elliptical or spindle-shaped, curved or slightly indented nucleus. This produces a nuclear outline described as a “footprint,” “C-shape,” “V-shape,” or “boomerang shape” (Fig. 12.4). The number of epithelioid histiocytes in a granuloma varies from 5 to 50 or more, and they typically appear as a syncytium. Figure 12.4 Sarcoidosis. Infectious organisms must be excluded by special stains and/or microbiologic culture results, but most fungal and mycobacterial infections are not purely granulomatous; rather, they are accompanied by necrotic debris and a neutrophilic, lymphocytic, and/or plasmacytic infiltrate. The cells of spindle cell sarcoma, although often in aggregates, have atypical nuclei with coarsely textured chromatin, and footprint-shaped nuclei are uncommon in spindle cell tumors. The dendritic cells in dendritic-lymphocytic aggregates have round to oval nuclei rather than elongated, curved nuclei, and germinal center elements (centrocytes, centroblasts, and tingible-body macrophages) are usually seen in assoociation. Granulomas accompany some malignancies (e.g., squamous cell carcinoma, Hodgkin lymphoma, T-cell lymphoma, seminoma) more often than others (adenocarcinoma, sarcoma). This is generally not problematic because the malignant cells are clearly seen and distinct from the granulomas. Infrequently, however, the granulomatous response is so pronounced it obscures malignant cells, or the malignant cells resemble epithelioid histiocytes.54 Aspirates of acute bacterial lymphadenitis are very cellular and composed of nearly a pure population of variably degenerated neutrophils. Pus is often seen in the hub or barrel of the syringe at the time of the procedure. One should submit a part of any purulent biopsy for culture, expelling it into a sterile container or culturette device. This should be done even if a patient is receiving antibiotics, because the organisms may be resistant to the patient’s current antibiotic therapy. In some examples, the organisms are seen directly on the Romanowsky-stained smear (Fig. 12.5). Figure 12.5 Acute lymphadenitis. In both bacterial and fungal lymphadenitis, identifying an organism by morphology or culture is important for more than just antibiotic selection, because the identification of an infectious agent aids in exclusion of a neoplastic cause for the necrosis and inflammatory reaction in an FNA specimen. Smears show numerous neutrophils and variably sized granulomas55 (Fig. 12.6). If only small granulomas are present, they may be obscured by the abundant neutrophils. The causative agent, the Gram-negative bacillus Bartonella henselae, may be stained using the Steiner method, but results are highly variable.56 Unless the organism is identified or isolated, the cytologic diagnosis is presumptive, not definitive, but serologic studies can be helpful. Figure 12.6 Cat scratch disease. Other organisms can produce an identical cytologic picture including Franciscella tularensis, which causes tularemia, Chlamydia trachomatis, which causes lymphogranuloma venereum, Yersinia enterocolitica or Y. pseudotuberculosis, certain fungi, and less commonly Mycobacterium tuberculosis. In most cases, microbiologic and/or serologic analysis are required for specific diagnosis. Early stages of cat scratch disease display very few granulomas and mimic reactive lymphoid hyperplasia.55 Mycobacterial infections occur in immunocompetent and immunosuppressed individuals. FNA is particularly efficacious in those countries where mycobacterial infection is endemic, but its accuracy may be lower in the United States because of the lower prevalence of disease, atypical clinical presentations,57 and better treatment of HIV-infected patients (who would otherwise consistitute the majority of patients with mycobacterial infections). Smears may show granulomas with necrosis, granulomas without necrosis, or sometimes necrosis only58 (Fig.12.7A). In immunocompromised patients, there may be only loose aggregates of histiocytes rather than true granulomas. Aspirates from patients with nontuberculous mycobacterial infection (typically Mycobacterium avium complex) may show the “negative image” phenomenon,59 which occurs because the lipid coat of the bacilli resists staining with any Romanowsky stain; thus, the bacilli are seen as optically clear rods/striations surrounded by stained proteinaceous or necrotic material. These rods may be extracellular or within macrophages, where they appear as multiple linear striations resembling the crinkled tissue paper appearance of the storage cells of Gaucher disease. This phenomenon is not visible with the Papanicolaou stain. Figure 12.7 Mycobacterial lymphadenitis. Cases of mycobacterial lymphadenitis with little or no necrosis resemble sarcoidosis. Special stains for bacteria, acid-fast bacilli, and fungi are important whenever granulomas, necrosis, and/or a neutrophilic infiltrate is present. Importantly, organisms are best seen with special stains in necrotic material (Fig. 12.7B), but the sensitivity of acid-fast bacilli stains is low, especially for M. tuberculosis. Molecular techniques, however, can detect and speciate mycobacteria. Alternatively, if necrosis or granulomas are seen at the time of the rapid evaluation of specimen adequacy, a portion of the needle rinse can be submitted for microbiologic cultures. Small lymphocytes vastly outnumber transformed lymphocytes, plasmacytoid lymphocytes, and immunoblasts. The histiocytes are large, with large pale nuclei, nucleoli, and abundant vacuolated cytoplasm; they can simulate the cells of an epithelial neoplasm. The key cytomorphologic feature is emperipolesis, the engulfment of lymphocytes by histiocytes (Fig. 12.8), but it may be difficult to appreciate, because engulfed lymphocytes can be so numerous that they obscure the underlying histiocyte nucleus.60,61 The large histiocytes are immunoreactive for S-100 protein and CD68. Figure 12.8 Rosai-Dorfman disease (RDD). Dendritic-lymphocytic aggregates in reactive lymphoid hyperplasia mimic histiocytes with emperipolesis. Instead of being engulfed by histiocytes, however, lymphocytes overlie and commingle with them in reactive hyperplasia. Since many of the lymphocytes “spill” from the cytoplasm as a consequence of smearing in RDD, they are often not strictly within the cytoplasm, but adjacent to it, thus adding to the confusion. An additional helpful feature is that histiocytes are much more numerous in RDD than in a reactive lymph node. Also, the histiocytes in dendritic-lymphocytic aggregates are negative for S-100 protein. Smears from patients with KL have a characteristic appearance that permits diagnosis by FNA in the proper clinical context.62 Granular proteinaceous and nuclear debris is admixed with distinctive histiocytes with a contorted, sharply angulated, often cresent-shaped, peripherally placed nucleus62,63 (Fig. 12.9). These histiocytes contain karyorrhectic debris but are readily distinguished from tingible-body macrophages, which are larger and have round nuclei. Plasmacytoid monocytes—medium-sized cells with an eccentrically placed round nucleus, condensed chromatin, and a moderate amount of cytoplasm—are also present, as are immunoblasts. Neutrophils are sparse or absent. No lymphocytic emperipolesis is present. Figure 12.9 Kikuchi lymphadenitis (KL). The combination of necrosis and contorted small phagocytic histiocytes, in the absence of neutrophils, is very striking. The characteristic small, contorted phagocytic histiocytes are easily overlooked on casual examination, but they are highly characteristic of KL and only rarely occur in other conditions.62 These cells are usually numerous and easily found if specifically sought, and, in the proper clinical context, the FNA report can strongly suggest the diagnosis of KL because the small phagocytic histiocytes are morphologically distinguishable from the larger tingible-body macrophages, which typically have round, centrally placed nuclei. Small phagocytic histiocytes can been seen in tuberculous lymphadenitis,62 a diagnosis that should be excluded with the help of histochemical stains and microbiologic cultures. A variety of bacterial and fungal infections can result in a necrotizing, suppurative, and granulomatous lymphadenitis, but a suppurative lymphadenitis contains abundant neutrophils which are not seen KL. The epithelioid histiocytes of a granulomatous lymphadenitis have elliptical, indented nuclei with smooth, rounded contours that are distinct from the angulated edges of the histiocytic nuclei of KL, and epithelioid histiocytes do not contain karyorrhectic debris. Lupus lymphadenitis is morphologically similar to KL,64 but smears from lupus lymphadenitis may contain hematoxylin bodies—darkly stained clumps of nuclear debris measuring 1 to 100 μm in diameter that have not been described in patients with KL.64 Even if hematoxylin bodies are absent from smears, it is prudent to exclude lupus with serologic testing whenever the FNA findings suggest KL. Aspirates contain predominantly small lymphocytes, accompanied by an increased number of immunoblasts, centroblasts, plasmacytoid lymphocytes, and occasionally plasma cells65,66 (Fig. 12.10). Binucleated immunoblasts simulating Reed-Sternberg cells may be found but are rare. Immunophenotyping confirms the polyclonal nature of the B-cells and often shows a reversed CD4/CD8 ratio. Figure 12.10 Infectious mononucleosis. A high percentage of immunoblasts, centroblasts, and plasmacytoid lymphocytes is typical of infectious mononucleosis, but the amount depends on the stage of the disease. In the early stages of infectious mononucleosis, immunoblast proliferation is barely perceptible, and FNA is indistinguishable from reactive lymphoid hyperplasia.66 The immunoblast proliferation in other lymphadenopathies (e.g., anticonvulsant-associated lymphadenopathy, herpes simplex, cytomegalovirus (CMV), and postvaccinial lymphadenitis) and drug hypersensitivity is indistinguishable from that of infectious mononucleosis. A detailed clinical history, serologic studies, or excisional biopsy may be necessary to exclude these other entities. Most patients who develop anticonvulsant-related lymphadenopathy (principally to phenytoin) usually do so within 6 months of the onset of therapy. Herpetic lymphadenitis usually occurs in the setting of disseminated infection in an immunosuppressed patient, and diagnosis is usually made by association with the cutaneous lesions. In addition to increased immunoblasts, herpes simplex and CMV lymphadenitis may contain cells with diagnostic viral inclusions.
Lymph Nodes
Indications for lymph node fine-needle aspiration
Limitations of lymph node fine-needle aspiration
Technical Aspects
Reporting Terminology and Accuracy
Ancillary Studies
Flow cytometry
Immunocytochemistry (cell block)
Morphology
lost
preserved
Sensitivity
high
lower
Specimen requirement
few cells, fresh (unfixed)
many cells, fixed
Detection of small monoclonal population
good
poor
Multiple labeling
easy
laborious
Hodgkin lymphoma
useless
useful
Turnaround time
2 hours
24–48 hours
Flow Cytometry
Immunocytochemistry
CD3
T cells
CD5
T cells, coexpressed in some B-cell lymphomas (small lymphocytic lymphoma and mantle cell lymphoma)
CD19
B cells
CD20
B cells
CD10
follicular lymphoma
bcl-6
follicular center cells/follicular lymphoma
CD23
small lymphocytic lymphoma (negative in mantle cell lymphoma)
PAX5/BSAP
B cells (recurrent/rituximab treated patients)
CD45
most lymphoid cells
κ
B-cell immunoglobulin light chain (assessing clonality)
λ
B-cell immunoglobulin light chain (assessing clonality)
cyclin D1
Mantle cell lymphoma
Ki67
proliferative rate (for grading)
CD15 (LeuM1)
Reed-Sternberg cells (except nodular lymphocyte predominant Hodgkin lymphoma)
CD30 (Ki-1)
Reed-Sternberg cells, anaplastic large cell lymphoma
Anaplastic lymphoma kinase
anaplastic large cell lymphoma
Molecular Genetic Studies
Non-Neoplastic Lesions
Reactive Hyperplasia (without Specific Etiology)
Immunoblasts and plasmacytoid lymphocytes are interspersed throughout a smear dominated by small, round lymphocytes, creating a polymorphous cell picture (Romanowsky stain).
A capillary emanates diagonally from this follicular center fragment (dendritic-lymphocytic aggregate), which contains a mixture of dendritic cells and a heterogeneous lymphocyte population (Romanowsky stain).
This dendritic-lymphocytic aggregate is a loose collection of small round lymphocytes and dendritic cells. The latter have pale nuclei with delicate cytoplasmic extensions (Papanicolaou stain).
Benign Lymphadenopathies with Specific Etiology
Inflammatory/Infectious Conditions with Characteristic Fine-Needle Aspiration Findings
Sarcoidosis
In sarcoidosis, the granulomas are tight aggregates of epithelioid histiocytes. The cells have oval or curved nuclei and abundant cytoplasm with indistinct borders (Romanowsky stain).
Bacterial and Fungal Lymphadenitis
Neutrophils are mixed with small, round lymphocytes. Note the chains of bacterial cocci (arrows) (Romanowsky stain).
Cat Scratch Disease
A discrete aggregate of epithelioid histiocytes is surrounded by large numbers of neutrophils (Romanowsky stain).
Mycobacterial Lymphadenitis
A, Necrotic material and only few degenerating nuclei (Romanowsky stain). B, A stain for acid-fast bacilli shows many extracellular bacilli and a macrophage filled with organisms (Ziehl-Neelsen stain).
Rosai-Dorfman disease (Sinus Histiocytosis with Massive Lymphadenopathy)
An enormous histiocyte has engulfed many small lymphocytes (Romanowsky stain). (Courtesy of Dr. Harry Kozakewich, Children’s Hospital Medical Center, Boston, MA, USA.)
Kikuchi Lymphadenitis
Histiocytes with contorted nuclei, including one with a C shape, contain ingested debris. These cells are significantly smaller than tingible-body macrophages (Romanowsky stain).
Infectious Mononucleosis
Many immunoblasts are mixed with small, round lymphocytes and plasmacytoid lymphocytes (Romanowsky stain).
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Lymph Nodes
Advantages of lymph node fine-needle aspiration
Ancillary studies help to
Advantages of flow cytometry
Advantages of immunocytochemistry
The amount of light scattered in different directions gives useful information about the cell
Commonly used fluorochromes, which emit pulses of different-colored light when excited by a laser beam
Advantages of using multicolor cytometry
Molecular techniques available
Two consistent findings in aspirated lymphoid tissue
Cytomorphology of reactive hyperplasia
Differential diagnosis of reactive hyperplasia
Lymphadenopathies that cannot be reliably distinguished from reactive lymphoid hyperplasia by fine-needle aspiration
Cytomorphology of sarcoidosis
Differential diagnosis of sarcoidosis
Cytomorphology of cat scratch disease
Differential diagnosis of cat scratch disease
Cytomorphology of mycobacterial lymphadenitis
Differential diagnosis of mycobacterial lymphadenitis
Cytomorphology of Rosai-Dorfman disease
Differential diagnosis of Rosai-Dorfman disease
Cytomorphology of Kikuchi lymphadenitis
Differential diagnosis of Kikuchi lymphadenitis
Cytomorphology of infectious mononucleosis
Differential diagnosis of infectious mononucleosis