Practical implications of the World Health Organization Reporting System for Lymph Node, Spleen, and Thymus Cytopathology: Categories and ancillary testing for subtyping of hematolymphoid tumors on FNA biopsy cytopathology using a pattern-based approach.

INTRODUCTION
The objective of the recently published World Health Organization (WHO) Reporting System for Lymph Node, Spleen, and Thymus Cytopathology

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(WHO system) is to encourage the global adoption of a standardized framework for the categorization of fine needle aspiration biopsy (FNAB) cytopathology of these hematopoietic sites, which will ultimately provide clinicians with well documented risks of malignancy (ROMs) for each category to support optimal diagnostic management decisions. This approach is also intended to facilitate the work of general cytopathologists in two key ways. First, assigning a case to one of a limited number of diagnostic categories simplifies reporting and makes interpretation of the report more straightforward. Second, by adhering to a global reporting system, both the cytopathologist and the referring clinician understand that each category carries a defined ROM, encompassing the possibility of false negatives and false positives. This review focuses on the categorization and diagnosis of hematolymphoid processes and neoplasms, rather than the well established and robust use of FNAB, to diagnose infections and metastatic lesions in lymph nodes.
The concept of analyzing FNAB smears of lymph nodes using a pattern approach has been previously published.
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The WHO system uses five categories covering all possible scenarios in FNAB of the lymph nodes, spleen, and thymus: inadequate/insufficient/nondiagnostic, benign, atypical, suspicious for malignancy, and malignant.
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The initial categorization is based on the cytopathology, most importantly correlated with clinical and ultrasound information, and can be further categorized after integrating clinical, imaging, cytomorphologic findings and results from ancillary techniques, when available. It cannot be overemphasized that cytomorphologic diagnoses, whether or not ancillary techniques are applied, are always made within a clinical context. This means that careful analysis of the clinical setting is mandatory before assigning a final diagnostic category and, when possible, establishing a definitive lymphoma subtype diagnosis. It goes without saying that relevant clinical information should always accompany the referral sheet.
For the nonexpert, the classification of hematolymphoid neoplasms may seem insurmountable, not least when based on cytopathologic specimens. To ease the task for the practicing cytopathologist, the WHO system has adopted a pattern‐based approach to facilitate analysis and narrow down the possible initial differential diagnoses.
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The following four patterns have been identified for hematolymphoid neoplasms: mixed lymphoid cell pattern; predominantly small/intermediate cell pattern; predominantly intermediate/large/pleomorphic/blastic cell pattern; and single, very large, atypical cell pattern (Table 1, Figures 1, 4, 7 and 11).
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In principle, the same pattern analysis can be applied to splenic or thymic hematolymphoid direct smears. Reactive histiocytosis, including granulomatous inflammation, histiocytic/dendritic cell neoplasms, metastases, and the rare primary mesenchymal tumors or carcinomas of the spleen and thymus, are considered separate patterns and are not further discussed here.

Insufficient/inadequate/nondiagnostic category
This category applies to cases in which the material obtained is insufficient for diagnostic evaluation because of either qualitative or quantitative limitations.
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Although there is no universally accepted definition of what constitutes an adequate preparation in lymph node FNAB cytopathology, the cytopathology report should clearly describe the features that led to the categorization as inadequate, insufficient, or nondiagnostic. The WHO system accepts that local usage will determine which of these terms a cytopathologist or institution uses but states that the single term selected should be adhered to and does not encourage the term nondiagnostic in hematopoietic FNAB cytopathology. A recent review of 10 retrospective and heterogeneous studies on the similar Sydney system revealed a wide range in the reported ROM of this insufficient category in FNAB from lymph nodes, varying from 17% to 67%.
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The authors and editors of the WHO system expect the overall percentage to decrease with improved FNAB techniques, use of key diagnostic criteria for reporting, and use of the structured categories.

Benign category
The benign category is used for those cases in which unequivocally benign features are present and may identify a specific process or lesion in a lymph node, the spleen, or the thymus.
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The ROM should be low and ranges from 2% to 16% in published studies.
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Atypical category
This category is used for those cases in which the cytopathologic features are predominantly benign but there are minimal features that raise the possibility of a malignant lesion. Commonly, the FNAB from a lymph node, spleen, or thymus has provided minimal material or the cytomorphologic features are partially or largely obscured because of poor smearing, staining, blood or ultrasound gel.
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The atypical category also includes cases that have technically adequate lymphoid material with largely benign cytopathologic features but in which the clinical setting, results from ancillary testing, and certain but limited cytopathology features raise the possibility of malignancy. The rational for creating this category is to underpin the specificity, negative predictive value, and low ROM of the benign category. The ROM for the atypical category is expected to be low or intermediate; however, a recent review of retrospective published studies has demonstrated a very wide range, from 29% to 77% reflecting different uses of the category, heterogeneous cohorts of patients, differences in clinical practice, and differences in the use of ancillary studies.
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Again, the use of the WHO system will assist in reducing these percentages.

Suspicious for malignancy category
The suspicious for malignancy category includes cases in which the material raises concern for hematolymphoid or metastatic malignancies. It is applied when cytopathologic features suggest malignancy but the sample is limited in quantity or lacks sufficient definitive characteristics to establish a conclusive malignant diagnosis.
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This often occurs in situations in which supportive clinical, immunophenotypic, or molecular data are insufficient or unavailable. This category supports the specificity, high positive predictive value, and high ROM of the malignant category while maintaining the high sensitivity for detection of malignant lesions with FNA. The ROM is expected to be high, consistent with data from published studies, which report rates ranging from 82% to 100%.
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Malignant category
A specimen classified as malignant exhibits unequivocal cytopathologic features indicative of malignancy.
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The published ROM is very high (range, 98%–100%).
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Ancillary testing
If resources permit, ancillary techniques, including immunocytochemistry (ICC), flow cytometry, in situ hybridization, polymerase chain reaction (PCR) analysis, and next‐generation sequencing, are helpful tools in the diagnostic work‐up of FNAB. Of these, flow cytometry is particularly valuable because of its broad applicability, rapid turnaround time, and ability to exclude or confirm many types of hematolymphoid tumors, often leading to a definitive diagnosis when correlated with clinical and cytopathologic findings without the need for histopathologic examination. The strength of flow cytometry resides in its ability to disclose monotypic B cells or T cells by virtue of expression of light chains (either kappa or lambda) or the constant regions of T‐cell receptor beta (TRBC; either TRBC1 or TRBC2) on discrete populations of B cells and T cells, respectively. Monotypia is suggestive of monoclonality, that is, the presence of a single clone with monoclonal B‐cell or T‐cell receptor gene rearrangement, but it is not equivalent to it.

Diagnostic approach to hematolymphoid neoplasms on cytopathology
Each of the four hematolymphoid patterns used in the WHO system raises benign and malignant differentials to consider. It is recognized that this approach is a simplification, subject to both individual interpretation and variability in disease presentation. For example, the spectrum of classic follicular lymphoma (cFL) cell types can produce a mixed lymphoid cell pattern and a predominantly small/intermediate cell pattern. Further, the size of the inflammatory component heavily impacts on the cytomorphological interpretation of lymphomas, particularly in smears with the mixed lymphoid cell pattern or the predominantly small/intermediate cell pattern. In some indolent B‐cell lymphomas, such as cFL and marginal zone lymphoma (MZL), it is not uncommon to find 10%–20% lymphoma cells or less in the FNAB material, as determined by flow cytometry. When as few as one in 10 or even one in five cells on the smear is lymphoma a cell, and when these cells are already difficult to distinguish from normal lymphoid cells on an individual basis, it becomes easy to understand why recognizing indolent B‐cell lymphomas can be challenging without ancillary techniques, particularly flow cytometry. Even with flow cytometry, in cases with many reactive T cells and few monotypic B cells, it may be very difficult or impossible to differentiate between monoclonal B‐cell lymphocytosis and indolent B‐cell lymphoma with a large inflammatory component. Usually, the clinical context will provide some clues, but the cytopathologist's choice between the atypical category (probably benign) and the suspicious for malignancy category (probably malignant) may be arbitrary. Inevitably, some false negatives and positives will be found here because only histopathology supported by the full range of ancillary tests will provide a reliable distinction in equivocal cases. Furthermore, limited involvement of a lymph node, thymus, or spleen may lead to misclassification. Hence, in rare cases, the mixed lymphoid cell pattern, the predominantly small/intermediate cell pattern, or even the single, very large, atypical cell pattern may be observed in high‐grade lymphoma if the involvement is partial.
One approach to maximize objectivity for the cytopathologist, if possible, is to first review the slides without knowledge of the clinical context or the results of ancillary testing, particularly flow cytometry, to avoid bias, and then to correlate with clinical and ancillary findings. Of course, this can be challenging for the interventional cytopathologist, who must integrate the real‐time cytomorphologic assessment at rapid on‐site assessment with clinical information and imaging findings to guide specimen triage and optimize diagnostic accuracy.
After categorizing the lesion into one of the four patterns, the clinical setting should be carefully reviewed to assess whether the lesion is more likely benign or malignant (Figures 1, 4, 7, and 11). If flow cytometry results are available, they often greatly aid in the final cytopathologic categorization and specific diagnosis (Figures 1, 4, 7, and 11). With these algorithms, all cases with adequate diagnostic material can be categorized as benign, atypical, suspicious for malignancy, or malignant. Of these, the vast majority (>80%) of lymphoid FNABs will be placed in the benign or malignant category, particularly if flow cytometry is used.
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In this context, it is important to bear in mind that the atypical category should be used when the cytopathologist is inclined toward a benign diagnosis but hesitates for some reason and should be used sparingly because the atypical category should not be a waste basket and is often the most difficult for clinicians to interpret. The WHO system accepts that not all FNABs can be definitive and provides a clear management approach to deciding what to do next when a case is categorized as atypical.
In a clinical situation that is regarded as likely to be benign, with benign or atypical cytomorphology, the absence of monotypic B cells or T cells by flow cytometry strongly argues in favor of a benign lesion (Figures 1, 4, 7 and 11). Conversely, in a clinical context likely to be malignant, with suspicious or malignant cytomorphology, the finding of monotypic B cells or T cells provides strong support for a malignant lesion Figures 1, 4, 7, and 11. Interpretation becomes less clear‐cut when a case is initially categorized as benign or atypical based on clinical and cytomorphologic assessment but monotypic B cells or T cells with specific markers of a particular lymphoma are identified by flow cytometry. In such cases, the cytopathology should be reviewed, and the suspicious for malignancy or malignant category needs to be considered, depending on the cytomorphologic pattern and the specific clinical context Figures 1, 4, 7, and 11. Difficulties in final categorization may also arise in cases for which there is clinical suspicion of malignancy but in the absence of clear‐cut cytopathologic evidence of malignancy and without evidence of monotypic B cells or T cells. Again, the interpretation and final WHO categorization will depend on the specific cytomorphologic pattern correlated with clinical and ancillary tests Figures 1, 4, 7, and 11.
The following is a detailed discussion of the four hematolymphoid patterns, the differential diagnoses associated with each pattern, the WHO system categories to consider, and the potential diagnostic pitfalls. In general, a definitive lymphoma subtype diagnosis is not possible without immunophenotyping, with the practical exception of recurrent disease or staging of a known hematolymphoid neoplasm. Nevertheless, using the full armamentarium of ancillary techniques allows for a definitive lymphoma diagnosis in the majority of cases (Table 1).

Mixed lymphoid cell pattern
The mixed pattern typically consists of a sizable fraction of small and intermediate lymphoid cells admixed with some large centroblasts from the germinal centers or immunoblasts with or without tingible body macrophages (Figure 2). If many eosinophils and plasma cells and histiocytes are present, the cytopathologic features are also likely to be interpreted as mixed (Figure 3).

Benign conditions
The mixed lymphoid cell pattern is typical of some common types of benign lymphadenopathy. In follicular hyperplasia, centroblasts and tingible body macrophages are easily found among small and intermediate‐sized lymphoid cells (Figure 2A). Flow cytometry will show polytypic B cells and T cells (Figure 2B). Immunoblastic reactions are characterized by a prominent population of immunoblasts along with small lymphocytes, plasmacytoid cells, histiocytes, occasional eosinophils, and occasional larger lymphoid cells.
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Prominent plasmacytosis is defined as an increased number of reactive plasma cells in a mixed lymphoid background.
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In all of these situations, flow cytometry is helpful for excluding the possibility of a hematolymphoid neoplasm. Polymorphic lymphoproliferative disorders arising in immune deficiency/dysregulation are characterized by a mixture of small lymphocytes, lymphoplasmacytoid cells, plasma cells, eosinophils, and large lymphoid cells (Figure 3A). By flow cytometry, monotypic B‐cells are sometimes found (Figure 3B). A definitive diagnosis of polymorphic lymphoproliferative disorder arising in immune deficiency/dysregulation based on cytopathology alone is not recommended, particularly when monotypic B cells are identified, because lymphoma cannot be reliably excluded without histopathologic confirmation.

Classic follicular lymphoma
cFL (formerly follicular lymphoma [FL] grade 1, 2, and 3A) is characterized by a mixture of centroblasts, centrocytes (intermediate lymphoid cells), and small lymphocytes, including B cells, T cells, and natural killer cells (Figure 2C).
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Lack of tingible body macrophages is typical, but the finding of occasional phagocytes does not exclude the diagnosis. The fraction of neoplastic cells may vary considerably; and, in certain cases, a prominent T‐cell–mediated inflammatory response may obscure the cytomorphologic identification of the lymphomatous centroblasts and centrocytes, and the pattern may be interpreted by some as the predominantly small/intermediate cell pattern. This is likely one of the reasons why cFL can be difficult to diagnose based on cytomorphology alone, particularly because the neoplastic centrocytes and centroblasts closely resemble their benign counterparts. Flow cytometry is highly effective in confirming cFL (Figure 2D) and, when combined with cytomorphology, can support a definitive diagnosis of FL in most cases (Table 1).
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Nevertheless, distinction from rare variants of FL, such as FL with a predominantly diffuse growth pattern, is not possible without a biopsy—preferably a surgical biopsy. To account for these limitations, it may be advisable to report cases consistent with cFL as FL, likely cFL.

Marginal zone lymphoma
MZL includes extranodal MZL of mucosa‐associated lymphoid tissue, splenic MZL, nodal MZL, pediatric nodal MZL, and primary cutaneous MZL.
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Of these, nodal MZL, pediatric nodal MZL, and splenic MZL are most likely to be encountered in FNABs from lymph nodes, whereas splenic MZL is the predominant differential diagnosis in the spleen. In all cases, cytomorphology resembles that of cFL, with centroblasts, intermediate lymphoid cells that have centrocyte‐like morphology, and small lymphocytes, except for the more frequent finding of tingible body macrophages indicative of the residual reactive germinal centers found on histopathology.
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Sometimes, lymphocytes resembling monocytes, monocytoid cells, can be found. By flow cytometry, the lymphoma cells are typically negative for CD5 and CD10. It is possible to make a definitive diagnosis of MZL on cytopathology combined with flow cytometry, provided that lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), CD10‐negative FL, and large B‐cell lymphomas have been excluded (Table 1).
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However, this may prove difficult in routine practice. Therefore, it may be advisable to sign out these cases as low‐grade B‐cell lymphoma, likely MZL, or possibly MZL, particularly if not all appropriate ancillary tests have been performed to rule out the differentials.

Nodal T‐follicular helper cell lymphoma, angioimmunoblastic type
Nodal T‐follicular helper cell lymphoma, angioimmunoblastic type, is characterized by a large inflammatory component, including lymphocytes, histiocytes, a few immunoblasts or Reed–Sternberg‐like cells, and usually some eosinophils and plasma cells.
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The neoplastic T cells often constitute a minor part of the cellular infiltrate and may be difficult to identify.
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Demonstration of monotypic T cells with a T‐follicular helper phenotype by flow cytometry can support the diagnosis of T‐cell lymphoma. However, a definitive diagnosis should not be made based solely on cytopathology and flow cytometry because other subtypes of peripheral T‐cell lymphomas may exhibit similar cytomorphologic and immunophenotypical features (Table 1).

Diagnostic considerations with the mixed lymphoid cell pattern
The mixed lymphoid cell pattern and the predominantly small/intermediate cell pattern are the most commonly observed patterns in routine FNAB practice; and, without flow cytometry, distinguishing between lymphoma and benign lymphadenopathy can be challenging, especially in clinically equivocal cases. When both the clinical context and cytomorphology raise suspicion for lymphoma, the identification of monotypic cells with other appropriate markers by flow cytometry supports the diagnosis, and the case can be categorized as Malignant and often signed out as a specific lymphoma (Figure 1, Table 1). Conversely, when suspicion for lymphoma is low, the absence of monotypic lymphocytes on flow cytometry supports a benign diagnosis, and the case can be categorized as benign (Figure 1). Diagnostic challenges may arise when there is a discrepancy between clinical/cytomorphologic assessment and flow cytometry findings. In cases with a low suspicion of lymphoma, the detection of monotypic lymphoid cells by flow cytometry may warrant categorization as atypical or suspicious for malignancy, depending on the broader clinical and cytomorphologic context (Figure 1). For example, in a clinical context and with cytomorphology suggestive of a polymorphic lymphoproliferative disorder arising in the setting of immune deficiency or dysregulation combined with the presence of monotypic B cells on flow cytometry (Figure 3), the atypical category may be the most appropriate classification.
cFL needs to be considered in cases that have an initial low suspicion of lymphoma on clinical and cytomorphologic grounds (Figure 1) but with skewing of kappa/lambda light chain by flow cytometry. Depending on the clone size, the immunophenotype, and light‐chain expression—lambda‐monotypic B cells are more likely to be malignant than kappa‐monotypic B cells—and considering that cFL typically exhibits dimmer expression of CD19, CD20, and CD38 compared with reactive hyperplasia,
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such cases may be appropriately classified as suspicious for malignancy or even malignant (Figure 1). On the other side of the spectrum, cases with an initial suspicion of malignancy on cytomorphology sometimes show polytypic expression of B and T lymphocytes. In such cases, the clinical context and the cytomorphology may need to be re‐evaluated because the absence of monotypic lymphocytes argues against lymphoma. This may support final categorization in the benign or atypical category (Figure 1). A typical clinical scenario would be a single prominent lymph node (>20 mm) in an adult patient without an obvious clinical infectious cause. Nevertheless, if clinical suspicion remains high, assigning the case to the suspicious for malignancy category may still be appropriate (Figure 1). In this context, it is important to emphasize that classic Hodgkin lymphoma (cHL) should always be considered when evaluating cases with a mixed lymphoid cell pattern. This represents one of the most common pitfalls in lymphoma cytopathology, and careless slide examination may result in missing a diagnosis of cHL.

Predominantly small/intermediate cell pattern
This pattern is composed of various proportions of small and intermediate cells that may or may not appear atypical (Figures 5 and 6). Centroblasts, immunoblasts, plasma cells, eosinophils, and tingible body macrophages are scarce.

Benign conditions
After resolution of lymphadenitis, an enlarged lymph node may persist long after the initial stimulus has subsided. Such nodes are a common reason for referral in routine clinical practice. This condition can be referred to as a resting lymph node or inert nonspecific lymphadenitis (Figure 4) because of the absence or minimal presence of germinal center activity and immunoblasts,
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The cytopathologic smear is dominated by small lymphocytes without centroblasts or tingible body macrophages (Figure 5A). It is typically diagnosed in patients with a single, persistent lymph node enlargement that neither progresses nor regresses over time.

Small lymphocytic lymphoma
Small lymphocytic lymphoma (SLL), which is the nodal counterpart of chronic lymphocytic leukemia (CLL), is characterized by a predominant population of small lymphoid cells. These cells may or may not exhibit atypical features and are typically accompanied by variable numbers of prolymphocytes and scattered paraimmunoblasts.
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Small cell morphology on cytopathology (Figure 5B), in combination with the characteristic immunophenotype (Figure 5C), is diagnostic of SLL, that is, CD19‐positive/CD20dim/CD5‐positive/CD23‐positive/CD200‐positive with dim monotypic expression of light chains (Table 1). Nevertheless, some caution may still be advisable because the immunophenotype may show some overlap with MCL. Therefore, it may be appropriate to sign out these cases as low‐grade B‐cell lymphoma, likely SLL/CLL if there remains some doubt, particularly if ancillary testing for cyclin D1, SOX11, or t(11;14) has not been performed.

Mantle cell lymphoma
A predominance of centrocyte‐like cells is characteristic of MCL (Figure 6A), although blastic and pleomorphic variants are well recognized. Scattered histiocytes are sometimes present.
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A conclusive diagnosis of MCL can be made on cytopathology, provided that nuclear staining for cyclin D1 can be demonstrated in the lymphoma cells (Figure 6B,C, Table 1). Some caution is necessary because the cells in the proliferation centers of SLL, which may exhibit an immunophenotype overlapping with MCL, can show nuclear cyclin D1 staining. Additional testing with SOX11 or fluorescence in situ hybridization (FISH) analysis may be necessary to confirm a diagnosis of MCL because cyclin D1‐negative cases are well recognized.
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Lymphoplasmacytic lymphoma
Lymphoplasmacytic lymphoma is characterized by a predominant population of small lymphoid cells that, to a variable extent, display lymphoplasmacytoid features and may include neoplastic plasma cells. Nuclear pseudoinclusions of immunoglobulins and round, dense cytoplasmic globules are common.
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Although most often encountered in bone marrow specimens, lymphoplasmacytic lymphoma can occasionally be seen in lymph node FNAB. On flow cytometry, the lymphoma cells are typically negative for CD5 and CD10. MZL is the main differential diagnosis. A definitive diagnosis on cytopathology is possible but may require PCR for detection of the MYD88 L265P mutation, along with clinical correlation, including immunoglobulin M paraprotein analysis (Table 1).

Plasma cell neoplasms
Plasma cell neoplasms are rarely encountered in lymph nodes. Distinction from benign plasma cell lesions can be achieved through flow cytometry or ICC by demonstrating an aberrant immunophenotype, such as CD56‐positive, CD117‐positive, and/or light‐chain restriction.
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Clinical correlation, including immunoglobulin G paraprotein analysis, can also be helpful (Table 1).

Mastocytosis
Mastocytosis is an uncommon finding in lymph nodes. In typical cases, numerous mast cells with pale, eccentric cytoplasm densely packed with numerous metachromatic granules (Giemsa stain) are identified. In some cases, mast cells with hypogranulated, polygonal, or spindle morphology predominate. Eosinophils, lymphocytes, histiocytes, and plasma cells may be present in the background.
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Aberrant mast cells expressing CD25, CD2, or CD30 can be readily detected by flow cytometry (with high sensitivity) or ICC, enabling a definitive diagnosis on cytopathologic specimens (Table 1).
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Diagnostic considerations with the predominantly small/intermediate cell pattern
The predominantly small/intermediate cell pattern is very common. Like with the mixed lymphoid cell pattern, flow cytometry is a powerful tool for excluding both B‐cell and T‐cell lymphomas, particularly when cytomorphology is equivocal. Many cases are straightforward and can be readily categorized as either benign or malignant, particularly after evaluation of flow cytometry results (Figure 4). Problems can arise when there are discrepancies between the clinical/cytomorphologic assessment and flow cytometry findings; for example, when monotypic B cells or T cells are detected in a case that appears benign based on both clinical and cytomorphologic evaluation. Depending on the size of the monotypic population, such cases may be categorized as atypical or suspicious for malignancy, or malignant: The atypical category may be considered when a small population (<0.5%) of monotypic B cells is present
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and monoclonal B‐cell lymphocytosis remains a differential diagnosis; however, if the proportion of monotypic B cells is substantial, the case may be categorized as suspicious for malignancy or even malignant, despite an initially low level of clinical suspicion for lymphoma. This scenario is particularly relevant in SLL, in which the clinical presentation may appear innocuous, and the neoplastic cells may look deceptively bland, closely resembling normal small lymphocytes, making cytopathologic diagnosis especially challenging, and most of these cases will be categorized as atypical and rely on flow cytometry to advance the diagnosis. At the opposite extreme, some cases may be clinically and cytomorphologically suspicious for a hematolymphoid neoplasm, yet no monotypic lymphoid cells are detected by flow cytometry. Obviously, plasma cell neoplasms and mastocytosis do not contain monotypic lymphoid cells detected by flow cytometry, but a cytomorphologically monotonous population of small lymphocytes may appear suspicious under the microscope and could be mistaken for low‐grade B‐cell lymphoma instead of the correct diagnosis of inert nonspecific lymphadenitis unless flow cytometry has been performed. Hence, depending on the clinical and cytomorphologic context, cases that are initially suspicious but lack monotypic lymphoid cells on flow cytometry may ultimately be categorized as benign, atypical, or suspicious for malignancy (Figure 4). Like the mixed lymphoid cell pattern, careful evaluation of all slides is essential to exclude the possibility of cHL, nodular lymphocyte‐predominant Hodgkin lymphoma (NLPHL), or T‐cell/histiocyte‐rich large B‐cell lymphoma (THRLBCL), all of which represent well known diagnostic pitfalls.

Predominantly intermediate/large/pleomorphic/blastic cell pattern
As the name suggests, this pattern is composed of a predominant population of intermediate or large cells that may be blastic or pleomorphic. Inflammatory cells, including lymphocytes, histocytes, plasma cells, and eosinophils, may be present in various proportions.

Benign conditions
A few benign conditions need to be considered with the predominantly intermediate/large/pleomorphic/blastic cell pattern (Figure 7). Kikuchi lymphadenitis and SLE‐related lymphadenitis have a similar cytomorphologic appearance. Both are characterized by many large lymphoid cells, sometimes including a necrotic background with karyorrhexis, which may give the impression of a high‐grade lesion (Figure 8A). Typically, some signet ring histiocytes are found (Figure 8A).
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Obviously, the clinical setting is extremely important for arriving at a correct conclusion. Flow cytometry reveals polytypic B cells and T cells (Figure 8B). Infectious mononucleosis can sometimes produce a worrisome clinical picture and cytomorphologic pattern, with numerous immunoblasts characterized by rounded nuclei with dispersed chromatin, one or two nucleoli, and dense basophilic cytoplasm (Giemsa stain), which may appear as a peripheral rim or be eccentrically located. Some immunoblasts may appear atypical and resemble binucleated or multinucleated Reed–Sternberg cells (RSCs). Flow cytometry is polyclonal, and the clinical situation usually gives important clues.

Lymphoblastic lymphoma
Lymphoblastic lymphoma is characterized by a predominant population of blasts with inconspicuous nucleoli.
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The diagnosis can be established by cytomorphology, with subtyping as B‐lymphoblastic or T‐lymphoblastic lymphoma established by flow cytometry. The typical phenotypes are CD19‐positive/CD79a‐positive/CD10‐positive/tdt‐positive/CD34‐positive or CD34‐negative, and CD3‐positive/CD7‐positive/CD10‐positive/tdt‐positive/CD99‐positive/CD1a‐positive or CD1a‐negative/CD34‐positive or CD34‐negative, respectively.

Large B‐cell lymphomas
There are multiple subtypes of large B‐cell lymphomas (LBLs), the most common of which is diffuse large B‐cell lymphoma (DLBCL), and the categorization as malignant can be made on cytopathology alone with a broad differential diagnosis of other malignancies, whereas subtyping requires ancillary testing. Others likely to be encountered in lymph nodes or spleen are DLBCL/high‐grade B‐cell lymphoma with MYC and BCL2 rearrangements and Epstein–Barr virus (EBV)‐positive DLBCL; or, in the thymus, primary mediastinal large B‐cell lymphoma.
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In many cases, a definitive subtype diagnosis can be made when the appropriate ancillary tools have been applied (Figure 9), provided that the large cells constitute more than 40% of all cells by both cytomorphology and flow cytometry (Table 1).
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This includes variants defined by genetic features, such as large B‐cell lymphoma with IRF4 rearrangement and high‐grade B‐cell lymphoma with 11q aberration,
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which can be readily identified by FISH or chromogenic in situ hybridization performed on FNAB smears or cell‐block preparations. Note that follicular large B‐cell lymphoma, (formerly grade 3b FL) and grade 3a cFL cannot be distinguished from DLBCL on cytopathology (Table 1).
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Burkitt lymphoma
Burkitt lymphoma (BL) typically displays many relatively uniform medium blasts with scant, deeply basophilic, and often finely vacuolated cytoplasm (Giemsa stain). The nuclei are round to mildly irregular with coarse chromatin and have two to four medium basophilic nucleoli.
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The categorization as malignant can be made on cytopathology with the relatively distinctive cytopathologic features at least suggesting a specific diagnosis, but the essential diagnostic features are reliably determined by flow cytometry (CD20‐positive/CD10‐positive/CD5‐negative), ICC (bcl2‐negative/bcl6‐positive), and FISH (IgH::MYC translocation), enabling a definitive diagnosis on cytopathology material (Table 1).
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Anaplastic large cell lymphoma
Anaplastic lymphoma kinase (ALK)‐positive and ALK‐negative anaplastic large cell lymphoma display similar cytomorphologic features that are categorized as malignant. Most cases are composed of large or very large cells that have doughnut, embryo‐like, or horseshoe‐shaped nuclei with prominent nucleoli and ample cytoplasm.
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A definitive diagnosis can be made on cytopathology material by demonstrating uniform, strong expression of CD30 using ICC on smears or cell blocks,
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along with evidence of monoclonal T‐cell populations (Table 1). This can be assessed by flow cytometry, using TRBC1 restriction analysis, or by T‐cell receptor gene rearrangement analysis using PCR.

Breast implant‐associated anaplastic large cell lymphoma
This is a mature T‐cell lymphoma composed of large cells with irregularly lobated nuclei, prominent nucleoli, and abundant cytoplasm and can be categorized as malignant on cytopathology. It typically arises in association with a breast implant, presenting as a transudate confined within a fibrous pseudocapsule. Less commonly, it may form a mass lesion that invades surrounding tissue.
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A definitive diagnosis can be established on cytopathology material by demonstrating uniform strong expression of CD30 by flow cytometry or ICC on smears or cell blocks (Table 1). Histopathology of the en bloc resected pseudocapsule and adjacent tissue is necessary for staging.
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Primary effusion lymphoma
Primary effusion lymphoma is a large B‐cell lymphoma presenting as a pleural, pericardial, and/or peritoneal serous effusion in the absence of lymph node involvement.
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The effusion contains numerous large cells with moderate‐to‐abundant, deeply basophilic (Giemsa stain) cytoplasm that sometimes contains vacuoles. The nuclei may be round or irregular and multilobated, often containing large nucleoli. The effusion can be categorized as malignant on cytopathology. By flow cytometry, the cells are CD45dim/CD38‐positive/CD138‐positive but negative for surface and cytoplasmic light chains and often for B‐cell antigens. Kaposi sarcoma–associated herpesvirus/human herpesvirus 8 infection is the sine qua non for diagnosis, which can be demonstrated by PCR (Table 1).

Peripheral T‐cell lymphoma, not otherwise specified
Peripheral T‐cell lymphoma (PTCL), not otherwise specified (PTCL‐NOS), represents a heterogeneous group of nodal and extranodal mature T‐cell lymphomas that do not fit into any defined PTCL subtype. It is characterized by a broad cytomorphologic spectrum, typically featuring medium‐to‐large, atypical lymphoid cells with varying degrees of nuclear pleomorphism that often can be categorized as suspicious for malignancy or malignant on cytopathology. Assessment of monoclonality by flow cytometry using TRBC1 or by PCR analysis of T‐cell receptor genes is often helpful in establishing a diagnosis of PTCL. Most PTCL‐NOS cases are CD4‐positive/CD8‐negative, although some may be CD4‐negative/CD8‐positive.
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Consequently, most PTCL‐NOS specimens cannot be reliably specifically diagnosed by cytopathology and ancillary techniques alone (Table 1). For example, nodal T‐follicular helper cell lymphoma, angioimmunoblastic type, requires histopathologic material (preferably an excision biopsy) to assess cell types, dendritic cell mesh works, and vascular proliferation. However, certain subtypes may be diagnosable by cytopathology, such as adult T‐cell leukemia/lymphoma (with serologic confirmation of HTLV‐1 infection) and primary EBV‐positive nodal T‐cell and natural killer‐cell lymphoma, which typically expresses CD8 and is positive for EBV–encoded small RNA.

Myeloid sarcoma
Myeloid sarcoma (MS) is a tumor mass occurring at a site other than the bone marrow, such as a lymph node, and is composed of myeloid blasts with or without evidence of maturation.
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The blasts are intermediate or large and have round‐to‐oval nuclei with blastic fine chromatin and inconspicuous or prominent nucleoli. The cytoplasm is scant to moderate, with a pale blue to blue‐grey hue (Giemsa stain). The diagnosis is usually straightforward when cytopathology is combined with flow cytometry or ICC, demonstrating myeloid blasts that typically express CD34 and/or CD117 (Table 1).

Diagnostic considerations with the predominantly intermediate/large/pleomorphic/ blastic cell pattern
This pattern is highly suggestive of a malignant lesion and most cases are straightforward to categorize as malignant, with or without the support of flow cytometry, especially when the clinical context raises suspicion for a hematolymphoid neoplasm (Figure 7). Importantly, some benign conditions, such as Kikuchi disease, SLE‐related lymphadenitis, and mononucleosis, may exhibit this pattern (Figures 7 and 8A). Although flow cytometry can aid in diagnosis (Figure 8B), it is well recognized that its reliability is reduced in this context because the cells in high‐grade lesions may be fragile and escape detection. Hence, these high‐grade lymphomas (Figure 10A) can usually be categorized as malignant even in the absence of monotypic cells by flow cytometry (Figure 10B).
Conversely, in a case initially categorized as benign or atypical, for example, an erroneous interpretation as infectious mononucleosis, the identification of large monotypic cells by flow cytometry supports a diagnosis of malignancy, particularly when the cellular pattern is predominantly intermediate/large/pleomorphic/blastic (Figure 7). A potential pitfall is the occasional presence of a granulomatous reaction within a high‐grade lesion, which may obscure malignant features and mislead the cytopathologist, particularly if flow cytometry is unavailable. Finally, metastases may need to be considered in the differential diagnosis of this pattern.

Single very large, atypical cell pattern
In this pattern, scattered, very large, atypical cells, which are alien in the sense that they do not resemble any lymphoid cell,
2
are found against a prominent inflammatory background, including lymphocytes, histocytes, plasma cells, or eosinophils in various proportions.

Benign conditions
A few benign conditions may produce the single, very large, atypical cell pattern, including: Warthin–Finkeldey lymphadenitis, characterized by scattered polykaryocytes, which are very large multinucleated cells with oval nuclei, fine chromatin, and often prominent nucleoli (Figure 12A); and Rosai–Dorfman disease, which exhibits scattered, large, sometimes multinucleated cells with round nuclei, prominent nucleoli, and abundant pale‐to‐eosinophilic cytoplasm frequently exhibiting emperipolesis with lymphocytes and other inflammatory cells in their cytoplasm. There is a mixed inflammatory background containing numerous lymphocytes, plasma cells, and neutrophils.

Classic Hodgkin lymphoma
cHL is among the most frequently missed initial diagnoses on cytopathology because of the various and sometimes sparse numbers of Hodgkin cells (HCs) and RSCs found in a background of small lymphocytes, variable numbers of histiocytes, and smaller numbers of eosinophils and plasma cells, depending on the subtype (Figure 12B).
1
RSCs are large cells with bilobed or two distinct but similar nuclei and prominent single or multiple, large nucleoli approaching the size of lymphocytes. HCs are smaller than RSCs and have single, round‐to‐more pleomorphic nuclei with one or more prominent nucleoli. A provisional diagnosis, but not subtyping, can be reached on cytopathology in many cases of nodular sclerosing and mixed cellularity correlating with clinical findings and can be confirmed by ICC performed on smears or cell blocks to demonstrate the typical phenotype, that is, CD30‐positive/CD15‐positive/pax5‐positive–weak (Figure 12B, Table 1).

Nodular lymphocyte‐predominant Hodgkin lymphoma
Nodular lymphocyte‐predominant Hodgkin lymphoma is characterized by scattered, very large cells with nuclear multilobation and one or more nucleoli (Figure 12C), often smaller than classic RSCs and with moderate, pale cytoplasm (Giemsa stain).
1
Unlike many cases of cHL, except the lymphocyte‐rich subtype, the inflammatory background contains small lymphocytes and histiocytes, usually without or with very few eosinophils and plasma cells (Figure 12C). The differential diagnoses include THRLBCL, which overlaps with NLPHL, cHL (particularly the lymphocyte‐rich subtype), and follicular hyperplasia. A definitive diagnosis on cytopathology is not possible, mainly because of the close resemblance to THRLBCL in both cytomorphology and immunophenotype. Histopathology, preferably surgical excision, is necessary to make the distinction (Table 1).

Lymphomatoid granulomatosis
Lymphomatoid granulomatosis is a rare, EBV‐driven B‐cell neoplasm with angiocentric growth, usually involving extranodal sites, composed of EBV‐positive, atypical, large B cells admixed with many reactive T cells.
9
Lymph node involvement is very rare. The role of FNAB is to demonstrate a mixture of large atypical lymphoid cells and reactive lymphocytes and to raise the differential diagnosis of lymphomatoid granulomatosis and other EBV‐positive lymphoproliferative disorders, including cHL, in the appropriate clinical setting.
1
A specific diagnosis cannot be made on cytomorphology because the diagnosis of lymphomatoid granulomatosis requires demonstration of angiocentricity of the lymphoid infiltrate (Table 1). If there is a uniform population of large, atypical, EBV‐positive B cells, EBV‐positive DLBCL should be considered.

T‐cell/histiocyte‐rich large B‐cell lymphoma
THRLBCL is characterized by scattered, large, atypical B‐lymphoid cells, comprising less than 10% of the infiltrate.
1
These cells may resemble the large lymphoma cells of NLPHL with multilobated nuclei, the large centroblast‐like or immunoblast‐like cells of large B‐cell lymphoma, or HCs and RSCs.
1
A definitive diagnosis should not be attempted on cytopathology and its material, not even with extensive flow cytometry, ICC, and/or molecular analysis, because histopathology on an excised node is necessary to exclude NLPHL and other differentials (Table 1).

Diagnostic considerations with the single, very large, atypical cell pattern
This pattern is highly suggestive of lymphoma, provided that the degree of atypia is sufficient (Figure 11) and that metastatic disease has been excluded. HCs should not be confused with histiocytes, dendritic cells, immunoblasts, or carcinoma cells (Figure 12B,C). A few benign conditions may present with this pattern, including Warthin–Finkeldey lymphadenitis (Figure 12A) and Rosai–Dorfman disease. However, the atypical cells in these lesions more closely resemble multinucleated giant cells (in Warthin–Finkeldey lymphadenitis) or large histiocytes (in Rosai–Dorfman disease) rather than malignant lymphoid cells. A polytypic result with flow cytometry means nothing because the scant malignant cells of cHL or THRLBCL usually escape detection by flow cytometry (Figure 11). Occasionally, monotypic cells may be identified, which, in this context, is more suggestive of a high‐grade lymphoma, either with a prominent inflammatory background or with only partial nodal, splenic, or thymic involvement. Therefore, the detection of monotypic large cells by flow cytometry, in conjunction with the single, very large, atypical cell pattern on cytopathology, should categorize the case as suspicious for malignancy or malignant regardless of the clinical context (Figure 11). Finally, it is important to remember that granulomatous reactions are not uncommon in cHL and may lead to an erroneous diagnosis.

CONCLUSION
Accurate cytopathologic categorization of lymphoid FNAB specimens using the WHO system can be achieved based on clinical information and the cytopathologic features of the wide range of reactive, lymphomatous, and metastatic lesions. More specifically, lymphoid lesions can be categorized and often diagnosed based on the cellular patterns presented in the WHO system, including the mixed lymphoid cell pattern, the predominantly small/intermediate cell pattern, the predominantly intermediate/large/pleomorphic/blastic cell pattern, and the single, very large, atypical cell pattern. Specific diagnoses of the lymphoid lesions do require careful integration of the clinical history, key diagnostic cytopathologic features (which are now defined in the WHO system by international consensus), and flow‐cytometric analysis in most malignant cases. In selected cases, additional studies, such as ICC, core‐needle biopsy, excision biopsy, or molecular testing, may be necessary. A thorough understanding of the full cytomorphologic spectrum of lymphoid and metastatic lesions and an awareness of common diagnostic pitfalls, particularly those involving T‐cell neoplasms and cHL, are essential to avoid misdiagnosis and ensure appropriate patient management.

AUTHOR CONTRIBUTIONS

Mats Ehinger: Conceptualization, writing—original draft, and writing—review and editing. Maria Calaminici: Writing—review and editing. Immacolata Cozzolino: Writing—review and editing. Pio Zeppa: Writing—review and editing. Andrew S. Field: Conceptualization and writing—review and editing.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.