Lymph Nodes : Cytomorphology and Flow Cytometry

Lineage-associated Markers

Antigenic markers of hematopoietic differentiation are called CDs (for clusters of differentiation). There are more than 350 CDs, but the ones used for clinical applications in hematopoietic malignancies are relatively limited. Still more limited is the number of CDs that can be tested in FNAs, because of the limitation of the material. Activation markers used in hematopoietic malignancies are CD38 and HLA-DR. The non-committed hematopoietic stem cells express CD34, and some of them express HLA-DR and CD38 in later stages. Another marker of immature cells is the terminal deoxynucleotidyl transferase (TdT) present predominately, but not exclusively, among lymphoid precursors.

Early on, the B-cell precursors express cytoplasmic CD22 and CD79, while surface CD19 and CD10 appear later. With increased differentiation there is a gradual decrease in CD10 and gradual gain in CD20. CD10, however, appears again in follicular center cells (FCCs) in the lymph nodes and the spleen. Normal mature B cells express surface immunoglobulin as well as CD19, CD20, CD22, and CD79b.

The lineage-specific marker for T cells is cytoplasmic CD3, which appears in the earliest T cells as precursors. Early T-cell precursors also express CD2 and CD7. During thymic maturation, the T-cell precursor undergoes rearrangements of the T-cell receptor. This process is accompanied by the appearance of other T-cell lineage-associated markers (CD5, CD4, CD8). The majority of the mature T cells are either CD4 ⁺ or CD8 ⁺ . Thymocytes, however, and aberrant neoplastic T cells may coexpress CD4 and CD8, as well as immature T-cell markers TdT and CD1a.

Cells of the myeloid lineage may be identified by the expression of CD13 and CD33, while CD117 and CD34 are expressed in immature and neoplastic myeloid precursors. The CD14 and CD11c are markers of monocytes. The most specific granulocytic marker is cytoplasmic myeloperoxidase.

All the above-discussed hematopoietic cells express the panhematopoietic marker CD45, but it is usually randomly expressed or negative in immature precursors, and plasma cells are CD45 ⁻ . Erythroid precursors also have downregulated CD45, but they have a high expression of CD71 (transferrin receptors) and later glycophorin A. CD56 is associated with natural killer (NK) lineage but may be expressed in some myeloid leukemias as well as multiple myelomas and plasmacytomas. Plasma cells express cytoplasmic immunoglobulins, bright CD38, and CD138. Large granular lymphocytes (LGLs) express CD57.

Sample Preparation and Processing

In our experience, effusions have to be washed several times, because many of them have nonspecific proteins attached to the cells. FNAs submitted in RPMI solutions can be washed only once or twice and then incubated with antibodies. However, if the aspirate is bloody, then a brief red blood cell lysis may be indicated. Following the red cell lysis (if needed), the cell yield is determined by automated or manual cell counts. Cytospins are made to serve as morphologic control and to ensure that the critical cells have been retained for analysis. They may also serve for future cytochemical or molecular genetic studies. The viability of cells can be assessed by FCM with 7-aminoactinomycin D (7-AAD) and dead cells are excluded from the analysis.

Test CD Panel Selection and Staining

Samples should be stained as soon as possible, to preserve viability and avoid clumping. The antibody panels chosen should be flexible and tailored according to the cytomorphology in the Diff-Quik examination and the patient's history. As will be seen in a later discussion, the most important is to assess for clonality, because B-cell lymphomas are the most common lymphoid neoplasm. Thus, if we have a small number of cells, 10 000 to around 200 000, we can select one tube that contains CD5, CD10, CD19, CD20, and kappa lambda light chains. If T-cell lymphoma is suspected, then we use CD3, CD4, CD8, CD5, CD2, and CD7 to start with. When the morphology is blastoid, then we use CD34 as well as the intracellular TdT in addition to CD3, CD19, and CD10. Permeabilizing agents are available commercially and can be used according to the manufacturers' instructions. In some laboratories, DNA ploidy and S phase are performed and can be useful in estimating the aggressiveness of lymphomas.

Data Acquisition

The light scatter measurement reflects the physical properties of the cells (cell size or internal complexities). The fluorescence data give information on the membrane and intracellular molecules, depending on the antibodies and dyes used for labeling the cells. The fluorescence signals are collected and amplified by photodetectors. The data for each of the cells are collected and stored in a list mode. The immunophenotypic data, as well as the physical characteristics (forward scatter, which reflects the size, and side scatter, which reflects the cytoplasmic complexity) are usually given as a dot plot pattern and analyzed. The analysis can be performed using the isotype control to exclude nonspecific fluorescence.

Analysis for Clonality

The most important function of FCM in cytopathology is to confirm the presence or absence of clonality. Since about 80–90% of lymphomas are mature B-cell neoplasms, the clonality can be assessed by surface kappa and lambda light chains together with CD19 and CD20 as B-cell markers. The significant kappa : lambda ratios quoted in the literature vary, but the normal is around 1.5 : 1. Generally, any case less or more than a ratio of 0.3–3 we view with suspicion, and examine carefully for an aberrant expression of other antigens or the presence of abnormal clones showing bright or dim expression of CD20 or CD19 (see below).

Pitfalls in Flow Cytometric Immunophenotyping

• About 15% of lymphomas may be negative for both kappa and lambda expression. The use of different commercially available kappa and lambda antibodies may overcome this problem. In our laboratory, in addition to a CD19 and 20 with kappa/lambda, we add another tube with kappa and lambda having reverse fluorochromes, so that we can compare the brightness and double check kappa/lambda expression. In addition, there may be obvious aberrancy-like coexpression of CD5 or CD10 in a B-cell population, which may turn out to be either kappa- or lambda-negative, or the presence of a brighter or dimmer CD19 or CD20 population.

• Some normal B cells may coexpress other antigens, for example CD5 or CD10, and care must be taken regarding their examination for clonality.

• Sometimes in certain conditions such as chronic lymphocytic leukemia (CLL), CD20 expression may be almost absent. With the use of anti-CD20 antibodies for treatment, many patients present with lymphomas showing no CD20 expression. Thus, examination of clonality preferably includes CD19 in addition to CD20.

• In up to 30% of LBCLs, the cells may not be detectable because they degenerate or disappear during processing, so a negative result may be obtained, and in these situations morphology must be taken in consideration.

• In some autoimmune conditions, for example Hashimoto's thyroiditis, and other reactive conditions, an oligoclonal B-cell population may be identified. Caution in making a definite lymphoma diagnosis and careful correlation with morphology and clinical findings are recommended in these settings.

• It is well recognized now that certain normal people, 3–5%, especially those older than 65 years, may have monoclonal B cells in the blood. These cells may be present in FNAs and will be recorded as minimal clonal populations. Thus, interpretation of a minimal clonal population must be undertaken carefully and in some cases, compared with the peripheral blood FCM. This principle is also true for body fluids in the diagnosis of minimal clonal population in effusions and even the cerebrospinal fluid if contaminated with blood.

• Clonality of T cells is difficult to prove by FCM alone, because, in certain situations, there may be abundance of CD4 ⁺ T cells. However, multiple deletions or losses of certain T-cell markers, such as CD3, CD5, or especially CD7, are common in T-cell lymphomas. Coexpression or absence of CD4 and CD8 expression are usually abnormal.

• Evaluation of cell size can be difficult, especially in FCC lymphoma in which centrocytes, which are a part of the “low-grade” component, can be large. Generally, in B-cell neoplasms we consider the events that are larger than the T cells, as potentially large cells, especially if clustered in one area of the forward scatter.

Immune Diseases

A variety of immunologic disorders are associated with lymphadenopathy, including rheumatoid arthritis, Sjögren's disease, systemic lupus erythematosus (SLE), and Kimura's disease. Most of the time, the lymph nodes are not biopsied because the clinical picture explains the etiology of the enlarged lymph nodes. However, unusual clinical situations may lead to a biopsy, as for example in cases of isolated lymphadenopathy or lymph node enlargement preceding the onset of disease, or in cases in which a lymph node reaches an unusually large size or grows at an unusually fast rate, raising the clinical suspicion of lymphoma.

The morphologic features of Sjögren's lymphadenopathy and rheumatoid lymphadenopathy may be indistinguishable with marked reactive follicular hyperplasia and plasmacytosis. FNA of these reactive hyperplasias is generally nonspecific, although immunophenotyping by FCM or even molecular analysis of immunoglobulin genes may be of extreme value in excluding the possibility of lymphoma. Kimura's disease, which is a benign chronic inflammatory disease of probable allergic etiology affecting predominantly Asians of young to middle age, differs by marked eosinophilic infiltration in addition to marked follicular hyperplasia. SLE also has nonspecific lymph node changes consisting of follicular hyperplasia and scattered plasma cells. Reports of necrotizing lymphadenitis dominated by individual cell necrosis and dispersed hematoxylin bodies or karyorrhectic nuclear debris may suggest the diagnosis of SLE, which can be confirmed by the appropriate clinical tests.

In some cases of autoimmune lymphadenitis, the hyperplasia can be extreme and associated with oligoclonal or even monoclonal B-cell proliferation. Attention to the clinical setting is important (see “ Flow Cytometry Overview ,” above).

Viral Infections

Post-vaccinial lymphadenitis and viral lymphadenitis accompanying a viral infection such as herpes simplex are associated with marked immunoblastic proliferation. Intranuclear viral inclusions may occur with certain viral infections such as herpes zoster lymphadenitis.

Epstein–Barr virus (EBV) is also the etiologic agent of classic infectious mononucleosis (IM), although other agents may be involved in atypical cases. IM is generally a self-limited infection most frequently occurring in adolescents and young adults. Manifestations include pharyngitis, fever, splenomegaly, peripheral atypical lymphocytosis, and lymphadenopathy. However, it is rare to see a lymph node aspirate from a patient with a typical clinical picture, because, in most cases, the presumptive clinical diagnosis is confirmed by examination of the peripheral blood and serologic evaluation. It is the atypical case, presenting with prolonged adenopathy or lymphadenopathy alone, that will prompt the clinician to perform lymph node sampling to rule out the possibility of malignant lymphoma.

Cytologically, lymph nodes affected by IM can be confused with malignant lymphoma because of the marked proliferation of immunoblast-like T cells. Aspiration biopsies yield markedly cellular smears with a heterogeneous lymphoid cell population that somewhat resembles follicular hyperplasia but with increased numbers of reactive lymphocytes at various stages ( Fig. 25-9 ). Some are large and often have single eccentric nuclei with smooth membranes, fine even vesicular chromatin, and a single large central nucleolus. The cytoplasm is often abundant and deeply basophilic and may contain a paranuclear “hof.” When binucleated, this cell may resemble a Reed–Sternberg cell. The differential diagnosis of IM includes other florid hyperplastic conditions, FCC lymphoma and LCLs, and Hodgkin lymphoma (HL). Immunophenotyping is very important in excluding malignancy. An abundance of CD8 ⁺ T cells is present in IM and a normal representation of the other T-cell antigens. B cells are uncommon. The appropriate serologic testing or in situ hybridization techniques can confirm the diagnosis of IM.

The most common cause of lymphadenopathy in acquired immune deficiency syndrome (AIDS) patients is reactive lymphoid hyperplasia. FNA is also useful in the diagnosis of tuberculosis and fungal infections such as Cryptococcus and histoplasmosis in these patients, as well as malignancy, including Kaposi's sarcoma and NHL associated with the immunosuppression of human immunodeficiency virus (HIV).

Suppurative Lymphadenitis

Pyogenic infections of lymph nodes, which are seen more commonly in children than in adult patients, are most often due to drainage of a regional bacterial infection. The lymph nodes in the head and neck region are the most frequently involved. FNA biopsies contain a predominance of intact and degenerated neutrophils. The lymphocytes that are present in the smears may also show prominent degenerative changes. Bacteria may be visualized, particularly on Romanowsky stains. On-site evaluation is important in these cases to triage material for microbial cultures.

Aspiration biopsy of cat scratch disease also yields numerous neutrophils in stellate abscesses containing peripherally palisading histiocytes and is therefore in the differential diagnosis of suppurative lymphadenitis ( Fig. 25-10 ). (This entity is discussed further under the heading of “Granulomatous lymphadenitis,” below.) Suppurative inflammation is also associated with actinomycosis, which is caused by Actinomyces , Gram-positive anaerobic filamentous bacteria. Actinomyces is a common saprophyte of the mouth and tonsils that can become pathogenic. Cervicofacial actinomycosis may appear as a mass simulating lymphadenopathy. The organisms are fairly conspicuous in aspirates as balls or aggregates of fine granules and radiating filaments with occasional branching surrounded by a marked acute inflammatory infiltrate with a few histiocytes and occasional lymphocytes ( Fig. 25-11 ).

Coccidioidomycosis caused by Coccidioides immitis is a fungal infection that incites an acute inflammatory reaction. C. immitis primarily infects the lungs but may disseminate to lymph nodes. In contrast to histoplasmosis that causes a granulomatous reaction (see below), aspirates of lymph nodes in cases of coccidioidomycosis are consistent with abscess formation. The endospore form of the organism can be found.

Granulomatous Lymphadenitis

Granulomatous lymphadenitis is a form of chronic inflammation characterized by granulomas formed by syncytial aggregates of epithelioid histiocytes with elongated nuclei, fine granular chromatin, and small nucleoli in a background of lymphocytes and occasionally plasma cells. There are generally two types of granulomatous inflammation: foreign body and immune granulomas. Foreign body granulomas are associated with inert foreign material, while the immune granulomas are caused by induction of a cell-mediated response to an insoluble particle that gets engulfed by macrophages. T lymphocytes get activated and cytokines such as interleukin-2 and interferon (IFN)-γ, which activate other T cells and macrophages, are produced. IFN-γ is the cytokine that transforms macrophages into epithelioid and multinucleated giant cells. Multinucleated giant cells consist of either the foreign body type (haphazardly arranged nuclei) or the Langhans type (peripherally arranged nuclei).

On Papanicolaou-stained preparations, the epithelioid cells tend to be spindly with wispy cytoplasm ( Fig. 25-12 ). Granulomatous inflammation is associated with a number of conditions including infections (classically fungi and mycobacteria), sarcoidosis, and foreign body and immune reactions. Certain malignancies, such as HL and seminoma, may be associated with a granulomatous inflammation, and a foreign body granulomatous reaction to keratin may be seen in well-differentiated squamous cell carcinoma.

Tuberculosis and leprosy are the mycobacterial infections that may affect lymph nodes. The granulomatous inflammation of tuberculous lymphadenitis is typically associated with necrotic material, although sometimes the epithelioid granulomas are without necrosis, particularly in the earlier stages of the infection. The term caseous necrosis is based on the gross appearance of the necrotic material, which looks cheesy. At times liquefied necrotic material with marked polymorpho­nuclear infiltration gives rise to suppurative lymphadenitis. Sometimes the aspirates may just contain the necrotic material without evidence of epithelioid granuloma. The number of lymphoid cells and giant cells is also variable. Various studies report wide differences in positivity for acid-fast bacilli stained by the Ziehl–Neelsen technique, with an average around 50% but ranging from 9% to 77%, with the greatest positivity occurring in suppurative lymphadenitis followed by caseous necrosis, and the least positivity in cases of epithelioid granulomas without necrosis. Definitive diagnosis can be established by polymerase chain reaction (PCR) on paraffin blocks.

The lepra bacilli of lepromatous leprosy are also acid-fast organisms that stain with the modified Ziehl–Neelsen technique. The classic cell of leprosy is the syncytial histiocyte (Virchow's cell or globus cell). These foamy, poorly circumscribed cells are frequently multinucleated with multiple coarse membrane-bound cytoplasmic vacuoles that tend to surround the nuclei. In the aspirate, the membrane-bound vacuoles are sometimes found lying free in the background, presumably resulting from rupture of histiocytes. The vacuoles are filled with numerous lepra bacilli. A range of cytologic findings can be seen in leprosy. In the tuberculoid form of leprosy, aspirates contain more lymphocytes and epithelioid type histiocytes, whereas in the lepromatous form of leprosy that is characterized by greater immunosuppression there are fewer well-formed granulomas and a more diffuse histiocytic infiltrate with few or no lymphocytes. The borderline stages between these two ends of the spectrum show a mixed cytologic picture reflecting the variation in the immune defect.

Natural or iatrogenic immunosuppression may lead to disseminated infection with other non-tuberculous mycobacteria, especially Mycobacterium avium-intracellulare . In immunocompromised patients, granulomas may be ill-defined and often consist of macrophages filled with acid-fast organisms, closely resembling what is seen in the immunocompromised form of leprosy. Smears made from heavily infected lymph nodes in immunocompromised states such as AIDS may be dominated by dispersed histiocytes with abundant pale-stained cytoplasm, which in Romanowsky stains may have a reticulated appearance due to numerous rod-shaped unstained phagocytized bacilli (negative images) ( Fig. 25-13 ).

Certain unusual bacteria are known for producing granulomatous lymphadenitis. Brucellosis is caused by the organism Brucella abortus , melitensis or suis and produces non-necrotizing granulomatous inflammation in lymph nodes. Infection is most commonly acquired through eating contaminated milk or cheese, resulting in follicular hyperplasia and loose clusters of epithelioid histiocytes. There may be a background of eosinophils, plasma cells, and immunoblasts that might be confused with HL. Cultures or molecular tests are needed to identify the organisms since they are otherwise not detectable in lymph nodes. Cat scratch disease is another bacterial infection that results in both suppurative and granulomatous inflammation in regional lymph nodes proximal to an inciting cat scratch. Close contact with a cat is cited by 90% of patients, and 75% of patients report being scratched by a cat. The agent of cat scratch disease is a coccobacillary extracellular organism, originally named Rochalimaea henselae , which is now classified as Bartonella henselae . In fully developed lesions, lymph nodes have follicular hyperplasia, granulomatous inflammation with stellate abscesses. Stellate abscesses have peripherally palisading histiocytes and central necrosis with numerous acute inflammatory cells ( Fig. 25-10 ). The organisms can be identified with a modified Steiner silver stain and are very difficult to culture. Serologic studies, immunofluorescence, or molecular methods rather than culture are better suited to confirm the presence of the organism.

Sarcoidosis is a systemic disease of unknown cause that is associated with non-necrotizing granulomas that can be seen in any organ. Bilateral hilar adenopathy is present in 90% of the cases, and transbronchial aspiration biopsy of hilar lymph nodes is often part of the workup. Lung parenchymal disease is the most common non-lymph node organ involved. Morphologically, sarcoidosis is characterized by numerous non-necrotizing granulomas with epithelioid histiocytes and giant cells. There is usually a background of reactive lymphocytes and scattered plasma cells. Sarcoidosis is a diagnosis of exclusion, but aspiration biopsies in the proper clinical setting containing prominent granulomatous inflammation without necrosis are very helpful in confirming the presence of sarcoidal inflammation in patients with lymphadenitis.

Sarcoidosis is seen more commonly in women and African-Americans, and the rates of disease are highest in the southeastern United States. The cause of sarcoidosis is unknown, but there is evidence that the disease involves immunologic abnormalities associated with CD4 ⁺ helper cells and their cell-mediated response to antigens. There is an expansion of the T-cell subsets and increased levels of T-cell cytokines, as well as increased levels of local cytokines that activate and recruit T cells and cause granuloma formation. There is also a genetic predisposition for sarcoidosis, with familial and racial clustering and association with HLA-A1 and HLA-B8. There is no evidence that sarcoidosis is caused by an infectious agent, but environmental causes have been proposed.

Berylliosis is a non-necrotizing granulomatous disease of the lungs often associated with bilateral hilar adenopathy that is caused by the inhalation of the toxic metal beryllium. The clinical presentation and cytomorphologic appearance of the granulomas is similar to that of sarcoidosis. Berylliosis was originally described in workers who made fluorescent light bulbs. Beryllium is now used in the aerospace industry and manufacturing of ceramic parts, and is associated with mining or refining of beryllium ores. A thorough occupational history is necessary to detect this disease.

Histoplasmosis is a common fungal infection affecting lymph nodes, often as a result of dissemination by the bloodstream after inhalation of spores or hyphae cause a primary pulmonary infection. In non-immune patients the fungus grows abundantly in macrophages and other phagocytic cells ( Fig. 25-14 ). In a resistant patient, Histoplasma excites an epithelioid granulomatous reaction that may or may not have necrosis. Histoplasma averages about 3 µm in diameter and has a rigid cell wall that is usually well stained by special stains for fungus such as Gomori methenamine silver. Aspiration biopsies of other disseminated fungal infections to lymph nodes appear similar to how they present in other organ sites.

Granulomatous lymphadenitis is also seen with the protozoan infections, toxoplasmosis and leishmaniasis. Toxoplasmosis is by far the most common in the USA, accounting for about 15% of all cases of unexplained lymphadenopathy. Animals and humans acquire infection by eating meat from chronically infected animals or by ingestion of oocysts deposited in soil, sand, or litter pans of cats. The cervical lymph nodes are most commonly involved but the adenopathy may be local or generalized, superficial, or deep (mesenteric, retroperitoneal, or mediastinal). Patients are most often asymptomatic but can present with fever, malaise, and atypical lymphocytosis, mimicking the clinical presentation of IM. Serologic tests for specific toxoplasma antibodies are the primary method of diagnosis; however, there are rare reports of FNA establishing a diagnosis of toxoplasmic lymphadenitis.

Characteristically, aspirates contain small and loose aggregates of epithelioid histiocytes with abundant cytoplasm and indistinct cell borders in a background of small and transformed lymphocytes and histiocytes containing phagocytized nuclear debris. In very rare reports, a toxoplasma cyst can be found in cytologic preparations. These cysts form during chronic infection, in which the crescent-shaped organisms, measuring approximately 2 by 6 µm, multiply slowly and are therefore called bradyzoites. They are tightly packed in “cysts” that originate in intracellular vacuoles that gradually enlarge beyond the usual size of the host cell, pushing the nucleus to the periphery and sometimes causing it to degenerate.

Leishmaniasis caused by the protozoan Leishmania prevails where climactic factors favor the propagation of sandflies, particularly South America, Africa, the Mediterranean basin, and Asia. After inoculation into the skin, the Leishmania (amastigotes) multiply within histiocytes that eventually rupture and release Leishmania , which enter other histiocytes. It may manifest as a cutaneous or visceral disease depending on the species of Leishmania involved. Rarely, Leishmania can present as a lymphadenitis alone without other clinical manifestations, and diagnosis by FNA has been reported. Lymph node aspiration biopsies contain a polymorphous population of lymphocytes, ranging from small lymphocytes to large plasma cells. The characteristic feature is the presence of epithelioid histiocytes with abundant cytoplasm filled with amastigotes ( Fig. 25-15 ). These are small ovoid or round cells measuring 1.5–3.0 µm in diameter with a thin cell membrane and a relatively large nucleus and a rod-shaped kinetoplast that is often lying tangentially or at right angles to the nucleus. These can be seen in Papanicolaou as well Romanowsky stains. Multinucleated giant cells and germinal center histiocytes are also frequently seen in smears.

Key Features of Granulomatous Lymphadenitis

• Syncytial aggregates of epithelioid histiocytes

• Multinucleated giant cells

• Background lymphocytes and occasional plasma cells

• Associated with infections, sarcoidosis, foreign body and immune reactions, and certain malignancies.

Two other parasites, the protozoan Trypanosoma gambiense and the filarial nematode Wucheria bancrofti , can present with lymphadenopathy in the early phase of the disease. These organisms are sometimes diagnosed by FNA, but they are not found within epithelioid histiocytes, as is the case for Leish­mania and Toxoplasma. Trypanosoma are elongate flattened spindle-shaped flagellated organisms with blunted posterior and pointed anterior ends. W. bancrofti are sheathed microfilaria with a pointed tail end free of nuclei ( Fig. 25-16 ).

Lymphomas Composed of Predominantly Small or Small- and Medium-sized Cells

One of the first steps in the morphologic evaluation of lymphomas is to assess cell size and nuclear features, resulting in a differential diagnosis of small-cell lymphomas versus LCLs. A process of elimination based on clinical, immunophenotypic, and molecular features further refines the differential diagnosis. See Fig. 25-17 for a simplified decision logarithm based on an initial evaluation of cell size.

In the process of lymphoma subclassification, it is important to be able to identify centrocytes, centroblasts, and other cellular components in the aspirate. The small-cell lymphomas often contain cells that resemble their normal counterparts (i.e., MZL, MCL). (For details, see “ Normal Lymph Node , ” above.)

Identification of centroblasts versus centrocytes is an important element in grading FL as well as predicting indolent from aggressive behavior. The majority of aggressive lymphomas have a centroblast count of ≥20%. Issues of interobserver variability and sampling make the count less reliable when in the range of 20–50%. The criteria, both histologic and cytologic, for transformation to LBCL are most clearly defined in FL (see below). In all types of small-cell lymphomas, particularly with borderline centroblast counts, clinical correlation is an essential part of the diagnostic process. Core needle and/or excisional biopsies may be necessary for primary diagnosis in most cases.

Low-grade Follicular Lymphoma

Follicular lymphoma represents about 20–40% of all NHLs in the USA, although there are geographic differences in incidence throughout the world. FL is predominantly a disease of older adults, with a median age at time of presentation of 55 years and approximately equal sex incidence. This type of lymphoma has been reported in children but with generally a more favorable outcome.

Follicular lymphoma is an indolent lymphoma with a reported 5-year overall survival of greater than 70%. However, with recent targeted therapies, the figure is probably much higher now. Patients usually present with lymphadenopathy involving multiple sites. Morphologic evidence of bone marrow involvement is reported in about 40% of patients. The disease is characterized by a slow progression with continuous and repeated relapses in spite of episodes of complete remission, with or without treatment. Most patients with advanced disease are incurable, although rare patients with limited-stage disease might be curable with radiotherapy. FL has a tendency to transform to a more aggressive lymphoma over time, most often LBCL at a rate of 3% per year. However, transformation to other entities can occur rarely, such as to BL, or to a composite lymphoma with large T-cell lymphoma. Transformation is usually associated with a poor prognosis, especially in heavily pretreated patients.

Cytomorphology and Ancillary Studies.

Follicular lymphoma is a monoclonal proliferation of B cells that have maintained the ability to recapitulate the follicular architecture of the normal secondary lymphoid follicle, at least focally within the tumor. There is a varying mix of small cleaved cells (small centrocytes), large cleaved cells (large centrocytes), and large or small nucleolated non-cleaved cells (centroblasts) similar to what is found in the follicular center ( Fig. 25-6 ). If a homogeneous population of lymphoid cells is present in an aspirate, FL would not be the first choice in the differential diagnosis on the basis of cytomorphology.

Relative lack of architecture is one limitation to the subclassification of FL by FNA. Some pathologists prefer to use the term follicular center cell lymphoma rather than follicular lymphoma for diagnosis on cytologic preparations in order to encompass the rare entity diffuse follicle center lymphoma, a rare variant of FL that has an entirely diffuse architecture. Attempts have been made to adapt histologic grading to cytologic preparations. WHO recommends a three-grade system based on the Berard method of counting the absolute number of centroblasts in 10 neoplastic follicles expressed per ×40 high-power microscopic fields (hpf). Grade 1 is defined as 0–5 centroblasts/hpf, grade 2 is 6–15 centroblasts/hpf, and grade 3 is >15 centroblasts/hpf. Grade 3 can be further subdivided into grade 3a and 3b, although to date the distinction between follicular grade 3a and 3b has not been shown to have clinical significance. In 3a, there are a number of centrocytes admixed with the centroblasts in the follicle center and it is often necessary to count the number of centroblasts to arrive at the >15 centroblasts/hpf cut-off, whereas grade 3b has solid sheets of centroblasts in the follicle center and is more obviously a grade 3 on first inspection.

Since FL appears to be a continuum from small cell-only infiltration to large cell-only infiltration, the cut-off between the three categories varies in different studies, with a rather subjective assessment by most pathologists even in surgical biopsies. For this reason, the two low grades, 1 and 2, are being combined as grade 1–2. The lack of architecture in aspirates presents even more of a challenge, since traditional grading is based on counting the number of centroblasts within neoplastic follicles. However, often overlooked is that follicular patterns can be readily appreciated in high-quality cellular smears ( Fig. 25-18 ). Sun and coworkers found an excellent correlation between grade and the percentage of large cells in smears of FL expressed as the percentage of large cells out of the total cell count (minimum total cell count of 200 up to 800 cells per case).

In practice, it can be difficult to distinguish every centrocyte from every centroblast. This accounts for some of the interobserver variability, for example, in grading FL. It can be helpful to identify large cells when they occur in aggregates according to the company they keep. For example, in a microscopic field heavily populated by centroblasts, most will be typically large, although some of the prominently nucleolated cells may appear smaller or more shrunken than others. While most of the centroblasts have prominent and sometimes multiple nucleoli, nucleoli may be more subtle in other large centroblasts with vesicular chromatin that are otherwise identical. Centroblasts tend to be more fragile and in some preparations may not be well preserved.

Even when follicular structures are not identified in aspirates, the overwhelming majority of aggressive lymphomas have a centroblast count of 20% or greater. However, issues of interobserver variability and sampling make the count less reliable when in the range of 20–50%. Furthermore, if one takes into consideration the dilutional effect of lymphocytes from peripheral blood in cytologic preparations, a lower number of large cells might be more significant than the same number in a tissue biopsy. Partial transformation to LBCL and grade 3 FL can also create an ambiguous mixture of small and large cells in aspirates, and in such circumstances a core biopsy is helpful and a surgical biopsy may be necessary.

Cell blocks are complementary to smears and provide additional architectural clues. The FNA process often aspirates follicular structures intact that can be appreciated in the cell block ( Fig. 25-19 ). One can count the percentage of centroblasts in these follicular centers but unfortunately there are generally at most only a few intact follicles in cell block preparations, not enough to follow the WHO system of counting centroblasts in 10 neoplastic follicles. However, cell blocks do give additional architectural clues, and when sheets of large cells are present ( Figs 25-20 , 25-21A–C ) they are as much a criterion for LBCL (follicular or diffuse) in aspirates as in surgical biopsies.

The vast majority of FCC lymphomas are CD10 ⁺ , CD5 ⁻ , and CD23 ^± monoclonal B-cell proliferations and are BCL6 ⁺ and usually BCL2 ⁺ ( Fig. 25-22 ). In about 90%, translocation (14;18) can be demonstrated by FISH but the test is usually not needed for diagnosis.

Primary Cutaneous Follicular Center Cell Lymphoma.

Lymphomas of the skin are usually biopsied rather than aspirated. Cutaneous lymphomas of germinal cell origin usually affect the head and neck of the elderly. They are usually CD10 ⁺ BCL6 ⁺ but almost always BCl2-negative. They can be follicular or diffuse and may have many centroblasts. Still, they have an excellent outcome.

Follicular Lymphoma “in situ” (Intrafollicular Neoplasia).

Sometimes lymphoid cells with germinal center immunophenotype and with the genetic and molecular features of follicular lymphoma, focally populate otherwise reactive follicles. This is to be differentiated from partial involvement of the lymph nodes by lymphoma. To our knowledge, there are no FNA studies of either entity, but extreme caution is recommended in the primary diagnosis of FL unless a biopsy is available.

Differential Diagnosis.

Aspirates of FL must be differentiated from benign reactive hyperplasia. Reactive follicular hyperplasia can be recognized in aspirates by the presence of a non-monotonous polyclonal mixture of small and large cleaved and non-cleaved FCCs. Sometimes, occasional plasma cells and scattered tingible body macrophages can be found. The BCL2 oncoprotein is expressed in about 80–90% of FLs, with less frequency in the large-cell subtype. In tissue sections, BCL2 immunohistochemistry stain can help differentiate benign follicular hyperplasia from FL. The neoplastic nodules are positive in FL and the pattern is reversed in reactive hyperplasia, with nodules being negative but surrounding small lymphocytes positive. In a cytologic preparation, the staining for BCL2 is not helpful because of the lack of architecture. However, if large centrocytes and centroblasts are positive this should raise the possibility of FL. Confirming monoclonality, most often by FCM, is the best way to preclude the diagnosis of benign reactive hyperplasia.

Follicular lymphoma must also be differentiated from other predominantly small or mixed small and LBCLs, particularly MCL, MZL, and small B-lymphocytic lymphoma ( Figs 25-23A, B , 25-24 ). Morphologically, FL tends to be less monotonous. They contain a mixture of small and large centrocytes as well as centroblasts, whereas MCL is composed of predominantly small irregular cells. MZL and Small lymphocyte lymphoma (SLL) tend to have more uniformly round or slightly irregular small- to medium-sized lymphoid cells with occasional immunoblasts. SLL tends to have very “blocky” or coarsely clumped chromatin. However, there is enough overlap of morphology between these entities that immunophenotyping is critical in differentiating the small- to medium-sized B-cell lymphomas.

SLL is both CD5 ⁺ and CD23 ⁺ ( Fig. 25-26 ). Aberrant coexpression of the T-cell marker CD5 is also the hallmark of most MCL, but in contrast to SLL it is usually CD23 ⁻ ( Fig. 25-27 ). The diagnosis of MCL can be confirmed by identifying the cyclin D1 protein by immunohistochemical staining of sections from cell blocks or by the demonstration of a 11;14 translocation by FISH. Marginal zone/MALT lymphoma is usually negative for CD5 and is CD10 ⁻ too; however, one must also consider the possibility of a CD10 ⁻ FL, a more common occurrence than a lymph node-based MZL. FISH analysis can be performed to detect a t(14;18) translocation involving the BCL2 locus that is characteristic of most FL if the distinction is clinically important.

Burkitt lymphoma and precursor B- or T-lymphoblastic lymphomas (LBLs) ( Fig. 25-28 ) may be composed of small- to medium-sized cells but unlike FLs are very aggressive. BL (which is also CD10 ⁺ BCL6 ⁺ ) consists of a relatively homogeneous population of medium-sized cells with round nuclei and coarse chromatin and several moderately prominent nucleoli. They have relatively abundant cytoplasm that contains small cytoplasmic vacuoles on Romanowsky smears. Precursor B or T LBLs are more monomorphic than FLs and may be rounded or convoluted depending on the variant. They typically lack conspicuous nucleoli and can be confused with a low-grade lymphoma for that reason. Most helpful in differentiation from FL is that LBLs are often of T-cell lineage and are almost always TdT-positive.

As stated previously, differentiating some cases of grade 2 FL from LBCL can be challenging in FNAs requiring counting of large cells, although interobserver variability remains a problem. Experienced clinicians can often use their clinical judgment in conjunction with the aspirate findings to guide their treatment decisions. However, a surgical biopsy is usually necessary for a definitive answer.

Key Features of Low-grade Follicular Lymphoma (Grades 1 and 2)

• Range of cell sizes

• Many centrocytes; and few centroblasts

• CD10 ⁺ , CD5 ⁻ B cells

• Light-chain restriction; about 15% light-chain-negative

• BCL6-positive; usually BCL2-positive

• t(14;18) usually present.

Marginal Zone/MALT Lymphoma

The normal marginal zone consists of the area just peripheral to the mantle zone of a secondary follicle. It is best appreciated in the spleen and is not easy to see in extra-abdominal lymph nodes. MZL is a low-grade B-cell lymphoid neoplasm that shows differentiation into either monocytoid B cells or plasma cells, theoretically similar to the differentiation of normal marginal zone B cells.

Marginal zone lymphomas may be nodal (uncommon) or extranodal. Extranodal MZL is also known as lymphoma of MALT, although it may occur in sites that are not usually thought of as mucosal-associated. Some low-grade MALT lymphomas may remain localized for a long time or have a long interval to relapse. At times, it may be difficult distinguishing benign lymphoid proliferations from early MALT lymphoma, such as those occurring in the bronchial or gastric mucosa. Cases of monoclonal B-cell proliferations in the stomach with microscopic features of MALT-type lymphoma can be cured by antimicrobial eradication of Helicobacter . In the stomach, but not in other anatomic sites, prominent lympho­epithelial lesions are features of MALT lymphoma. FNA would not be able to give this information, although most of these cases would be detected by endoscopic biopsy rather than cytologic methods.