Lymph Node Cytology: Morphology and Beyond

Abstract

Fine needle aspiration cytology (FNAC), being minimally invasive, rapid, cost-effective provides a valuable first-line diagnostic tool in the evaluation of lymphadenopathies both benign and malignant. Various ancillary techniques namely immunocytochemistry, flow cytometry, cell blocks, and molecular studies further improve the diagnostic accuracy of FNACs. Targeted FNAC under ultrasound guidance optimizes cellular yield in palpable and non-palpable lymphadenopathies. FNAC proves to be indispensable at establishing tissue diagnosis in cases when surgical excision is unfeasible, as in elderly patients with comorbidities or in metastatic settings. Nevertheless, lymph node FNAC represents a daunting task owing to the multitude of benign and malignant causes of lymphadenopathy. To aid categorization and better communication to the clinician, an emphasis on classification and reporting of lymph node cytopathology using Sydney system is laid upon.

1. Introduction

Lymph node enlargement can occur due to a variety of causes including benign, primary neoplastic, and metastatic. Fine-needle aspiration cytology (FNAC) provides a valuable tool in the diagnostic workup of lymphadenopathy due to a variety of causes namely reactive, infective, metastatic, and neoplastic. It is rapid, accurate, reliable, economic, safe, and more or less painless. FNAC can provide material for various ancillary investigations such as flow cytometry (FCM), immunocytochemistry (ICC), microbial culture, cell block, and molecular studies.

1.1 Normal anatomy and histology of lymph node

The lymph node (LN) is a bean-shaped, capsulated spongy sieve, which filters the lymph entering via the afferent lymphatics on the convex side and flowing out via the efferent lymphatics through the hilus [1]. The lymph node is a secondary lymphoid organ along with spleen, mucosal, and cutaneous lymphoid tissues. Thymus and bone marrow constitute the primary lymphoid organs [2].

LNs are aggregates present along the lymphatic channels. The LNs are situated at strategic sites in our body which helps in a quick and effective immune response [3]. The lymphocytes enter the LNs through blood or afferent lymphatics and get organized in T- and B-cell areas in the LN similarly, antigens enter the LN via afferent lymphatic channels, and are presented to the lymphocytes by dendritic cells (DC) which are the most efficient antigen-presenting cells (APC) [4].

The LN is divided into compartments by the connective tissue trabeculae which provide the framework for circulatory sinuses which are lined by the endothelial cells and divided into three regions: cortex, medulla, and paracortex [1]. Cortex is the area beneath the capsule and medulla is toward the hilus; cortex and medulla represent B-cell region. The paracortex is the area between cortex and medulla; represents T-cell region having a CD 4:CD 8 ratio of 3:1 along with presence of DC [1]. The cortex, depending on the activity of the LN, contains primary as well as secondary follicles. Primary follicles (unstimulated node) have small resting B cell aggregates and secondary follicles (stimulated LN) have an outer narrow mantle zone (containing B lymphocytes) and an inner germinal center (containing centrocytes and centroblasts) [5]. Table 1 highlights the antigen expression of B lymphoid cells according to the stage of maturation. Table 2 highlights the antigen expression according to their location.

B lymphoblast	CD 34, TdT, CD 19
B lymphoblast (pre-B cell)	CD 10, CD 19, CD 79a, PAX5, TdT
Immature B cell (attached with IgM)	CD 10, CD 19, CD 20, CD 79a, PAX5, TdT
Naive B cell (loss of TdT) (mantle cell zone)	CD 19, CD 20, CD 22, CD 79a, PAX5, CD 5+/−, CD 23

Table 1.

Immunohistochemistry (IHC) of B lymphocytes according to their maturation.

Sub capsular sinus histiocytes	CD 68, CD 163
Marginal zone	CD 5−, CD 10−, CD 20+, CD23−, CD 11c+, cyclin D1−, CD 103−
Mantle zone	CD 5−/+, CD 10−, CD 20+, CD 23−, CD 11c-, cyclin D1+, CD 103−
Germinal center	CD 10+, Bcl 6+; Centrocytes and centroblast (CD 10+, Bcl 6+); Helper T cell (CD 3, CD 4, CD5, CD7); Follicular DC (CD 21, CD 23, CD35); Tingible body macrophage

Table 2.

IHC of cells according to location in LN.

1.2 Approach to lymph node FNAC

1.2.1 Clinical history and examination

A prior detailed clinical history is required in each case to correlate the cytological findings. Patient’s age, duration of LN enlargement, history of fever, malaise, significant weight loss are all pertinent to be included. Any previous history of malignancy, surgery, or prolonged illness along with relevant blood and radiological investigations if available should be duly noted. The site, size, consistency, mobility, and number of LN involved should be carefully palpated and recorded. Any LN >2 cm is significant. Supraclavicular lymph nodes mostly harbor metastatic disease as compared to posterior cervical which is often reactive [6]. Soft, tender lymph nodes are seen in the inflammatory process while hard, fixed tend to be often malignant whereas rubbery consistency is suspicious of lymphoma.

1.2.2 Sample collection and cytologic preparation

Peripheral LNs can be easily palpable, thus FNAC of such LN is a simple procedure, which can be done blindly, preferably by a cytopathologist and does not require any local anesthesia. Site of FNAC is properly palpated, node fixed between two fingers of one hand and needling followed by aspiration done using the other hand. The needle used should be 22-24G and hand movement be brisk to avoid dilution of sample with blood since LNs are highly vascular structures (Figure 1). Once aspirated, the material should be checked for color and consistency as this provides a valuable information about the disease process. For example, infective lesions such as tuberculosis yield a thick purulent aspirate while that involved by lymphoma usually yield blood mixed aspirate. A necrotic aspirate is often seen in large metastatic LNs with areas of tumor necrosis. Aspirating only the cystic necrotic focus in such cases leads to a misdiagnosis if the firm areas containing viable tumor cells are missed. It is therefore important to completely aspirate the purulent material followed by needling of the firm areas. The sample obtained is gently spread over a glass slide with the help of another slide in a smearing fashion. Both air-dried and wet fixed smears are prepared and stained with routine stains such as May-Grunwald Giemsa (MGG), Papanicolaou (PAP) stain, and Hematoxylin& Eosin (H&E) stains. Extra smears can be air-dried and fixed with methanol for further usage. Additionally, cell blocks must be prepared for ancillary studies.

Figure 1.

Needle aspiration of lymph node by using 24-guage needle with plunger.

1.2.3 Ancillary techniques

Ancillary studies in LN aspirates include cell block preparation, special stains, immunocytochemistry (ICC), flowcytometry (FCM), molecular studies, and electron microscopy (EM). Adding to the advantages of FNAC, the ancillary studies help to arrive at a diagnosis, almost always, thus avoiding the need for an open biopsy. Cytology report when combined with the reports of ancillary techniques is well accepted by the clinicians and other cytopathologists [7].

It is, thus, advisable to always aspirate extra material which could be further helpful in performing the ancillary techniques. The cases are triaged based on examination of the MGG, PAP, and H&E-stained slides. In cases which are suspected to be infective, mycobacterial, or fungal in origin, special stains such as Ziehl-Neelson (ZN) stain and Periodic Acid Schiff (PAS) stain along with culture and PCR can be performed [5].

In cases of clinically suspected metastasis to the lymph nodes, cell blocks are prepared using various methods such as plasma thrombin method, fixed sediment method, scraping of cytology smears, histogel method, gelatin embedding method, colliding bag method, albumin method, bacterial agar method, and many more [8]. Cell blocks are micro biopsies embedded in paraffin section using the FNA technique. The residual clot or tissue in the hub of the needle is carefully removed and rinsed in 10% buffered formalin, centrifuged at 4000 rpm for 6 mins to form pellet. Supernatant is discarded and the sediment is placed on filter paper and processed as a routine histopathology specimen. Cell blocks are useful in better preservation of the morphology, architecture, and further help in performing IHC to find the primary [8, 9].

When evaluating a case of lymphoproliferative disorder, FCM and ICC are both helpful. FCM helps in immunologically characterizing the aspirated cells which are suspended in borate buffered saline (BBS) solution at pH 7.4. For complete characterization, cell concentration of approximately 1 million cells per ml buffer is required. FCM utilizes the principle of light scatter which measures the physical properties of a cell. Forward scatter (FS) depicts cell size and side scatter (SS) depicts cytoplasmic complexities. The fluorescence data is collected by a photodetector. Various antibody panels are used depending upon the cell morphology on giemsa stained smears.

In suspected cases of B-cell lymphoma antibodies which may be used are CD5, CD10, CD19, CD20, kappa, and lambda. In cases of suspected T-cell lymphoma CD 3, CD4, CD8, CD5, CD2, and CD7 are utilized. For immature blast identification TdT, CD34, HLA-DR, and CD38 are helpful markers. FCM provides the advantage of performing discrete measurements on millions of cells in a single sample.

FCM has a rapid turnaround time as compared to ICC, however, the morphology and staining characteristics are more preserved in ICC on cell blocks. Also, large cells, such as Reed-Sternberg (RS) cells, are better appreciated by ICC/IHC on cell blocks [9].

Transmission electron microscope (TEM) uses a fine electron beam, created by a tungsten filament heated by a high-voltage, electric current-heated and focused by magnetic lenses. The electron beam passes through an ultrathin plastic section in which tissue and cellular components have been post-fixed in osmium and impregnated with heavy metals such as uranyl and lead. A differential transmission of electrons occurs due to difference in densities of these heavy metals. An image is then captured on a fluorescent screen and viewed through a binocular microscope. It enables the visualization of details not apparent via light microscopy. TEM has an important role in cytopathology. Cells in effusions and FNAC samples can be readily prepared for TEM. It allows easy distinction of epithelial cells from mesothelial cells. They can be optimally fixed by directly injecting the aspirate into glutaraldehyde and treated as a cell suspension. As compared to IHC stains, TEM often is at least as specific and is also more economical in solving a diagnostic problem in specific conditions [10].

Molecular studies such as fluorescence in situ hybridization (FISH), Polymerase chain reaction (PCR), mutation analysis, laser-assisted microdissection (LMD), and DNA sequencing (classical and next-generation sequencing [NGS]) are valuable tools for the detection of genetic changes and hence provide improved early diagnosis of malignancy, prognostication, and prediction of response to therapy [11]. Detailed use of these studies will be discussed in subsequent sections.

FNAC from LN, benign or malignant, usually have a high cell count. The aspirates show lymphoid cells having fragile cytoplasm along with the presence of cytoplasmic fragments in the background called “lymphoglandular bodies,” measuring around 8 microns [1, 12]. They are round, small, pale and basophilic and their presence is important to identify the lymphoid origin of any lesion [1]. The cells seen in a normal LN lesion include mature lymphocytes (B or T phenotype), plasma cells, centrocytes (B cells, with cleaved nucleus), centroblasts (larger than centrocytes, marginal nucleoli), immunoblasts (largest of all lymphoid cells, with an eccentric round nucleoli) and macrophages [5].

2. Non-neoplastic lymphadenopathy

2.1 Reactive lymphoid hyperplasia

Reactive or follicular lymphoid hyperplasia is a common cause of lymphadenopathy, particularly in the younger age group and children. FNAC smears are highly cellular comprising a polymorphous population of small lymphocytes, follicular center cells (centrocytes and centroblasts), immunoblasts, plasma cells, dendritic cells, tingible body macrophages, occasional eosinophils, and polymorphs in a background of lymphoglandular bodies. It is divided into germinal center type, paracortical type, mixed type, and histiocytic reactions.

2.2 Granulomatous lymphadenitis

Granulomatous lymphadenitis is a chronic inflammatory response to foreign bodies or infectious agents. It is characterized by syncytial aggregates of epithelioid cells forming granulomas, varying number of multinucleated giant cells surrounded by small lymphocytes and plasma cells. Granulomas can be associated with necrosis (necrotizing granulomas) or be present without it (non-necrotizing/naked granulomas).

2.2.1 Tuberculosis

Tuberculosis is the commonest cause of granulomatous lymphadenitis in India. It is caused by infection by Mycobacterium tuberculosis. Tuberculous lymphadenitis is the most common manifestation of extrapulmonary tuberculosis and can be seen in any age group [7]. Patient usually presents with painless enlargement of LN accompanied with or without fever, cough, and weight loss.

Cytology smears show multiple epithelioid cell granulomas, Langhans type of multinucleated giant cells, and reactive lymphoid cells in a necrotic background. Epithelioid cells are histiocytes with characteristic elongated nuclei resembling the sole of a shoe [12]. The nuclear chromatin is fine, granular and the cytoplasm is abundant, pale, without distinct cell outlines. Multinucleated giant cells have abundant cytoplasm with multiple nuclei arranged peripherally in a horse-shoe shape pattern (Langhans giant cells). Necrosis seen in tuberculous lesion is characteristic caseous necrosis, getting its name from its cheesy appearance which on microscopy appears extracellular, eosinophilic, and granular in appearance. In cases where only finding is necrosis, demonstration of acid-fast bacilli (AFB) by Z-N staining or AFB culture helps clinch the diagnosis.

2.2.2 Sarcoidosis

Sarcoidosis is a disease of unknown etiology, seen more commonly in women and African Americans in the 3rd–4th decade [12]. It is associated with non-necrotizing granulomatous lymphadenitis and is often indistinguishable from other causes of granulomatous lymphadenitis, hence is a diagnosis of exclusion. Cytology smears show multiple non-caseating epithelioid cell granulomas, multinucleated giant cells, reactive lymphoid cells, and plasma cells. Calcium oxalate crystals and asteroid bodies may sometimes be seen in giant cells of sarcoidosis. However, no necrosis or any organism is demonstrable.

2.2.3 Leprosy

Leprosy, a systemic chronic infection caused by Mycobacterium leprae was completely eradicated in 2000 from 98 countries of the world, although it is found in underdeveloped countries with variable prevalence [13]. Cytology smears show clusters of foamy histiocytes formed by cells having multivacuolated bubbly cytoplasm, central nuclei, fine nuclear chromatin, and prominent nucleoli. Epithelioid cell granulomas and multinucleated giant cells may also be seen depending on the immune status of the patient. The air-dried Romanowsky stained smears may show presence of negative shadows representing the bacilli. When the bacillary load is high as in case of lepromatous leprosy, the cytoplasm of the histiocytes is filled with parallel arrangement of bacilli in the form of globi (Virchow’s/globus cell). Modified Z-N staining helps stain these bacilli.

2.2.4 Fungal lymphadenitis

Fungal lymphadenitis can be caused by a variety of fungal organisms such as Histoplasma capsulatum, Cryptococcus neoformans, Coccidioides immitis, etc. Cytology aspirates from fungal lymphadenitis are variable, having: (1) purely neutrophilic infiltrate, (2) only granuloma, (3) an admixture of the two, or (4) only demonstrate fungal organisms, with or without necrosis [7]. Histoplasma measures around 2–3 μm in diameter, has a rigid cell wall and resides in the cytoplasm of macrophages. C. neoformans measures around 5–10 μm and has a thick mucopolysaccharide capsule. Both demonstrate a narrow-based budding [7]. Histochemical stains such as methenamine silver, mucicarmine, and PAS along with fungal culture help confirm the diagnosis.

2.2.5 Foreign body granulomas

Granulomatous lymphadenitis can be elicited against foreign substances which are large enough to be removed by the body’s immune system. Some examples of the same include talc, silicone, suture material, or beryllium [7]. Cytology smears are mainly composed of giant cells containing foreign body particles, mature lymphocytes, and histiocytes.

2.2.6 Leishmania

Leishmania donovani is the causative agent of visceral leishmaniasis and is carried by sandfly which injects the promastigote form of leishmania into the skin. The promastigote form converts into amastigote form inside the histiocytes, which are released into the reticuloendothelial system (RES) upon rupture of the histiocyte. Cytology smears reveal amastigotes as oval structures of 1–3 μm size with a fine membrane, large nucleus, and a rod-shaped kinetoplast, present both intra and extracellularly admixed in a population of lymphocytes, histiocytes, and plasma cells [7]. Epithelioid cell granulomas and giant cells may also be seen occasionally.

2.2.7 Filaria

Wuchereria bancrofti, Brugia malayi, and Brugia timori are the commonest nematodes causing filariasis. Cytology smears can sometimes show microfilarial parasite in the LN aspirates. The microfilariae of both Wuchereria and Brugia are sheathed ranging in size from 200 to 300 μm in length, 2–8 μm in diameter, the former has a pointed tail that is free of nuclei and the latter has two distinct nuclei in the tip of the tail [14]. Smear may also show many eosinophils and epithelioid cell granulomas in the background.

2.2.8 Toxoplasma

Toxoplasmosisis a zoonotic disease caused by Toxoplasma gondii. Cytology smears show necrotic, polymorphous lymphoid background admixed with loose collections of epithelioid cell histiocytes and tingible body macrophages. Numerous small, elongated, crescent-shaped organisms may be seen [15].

2.3 Rosai Dorfman disease

Destombes first described Rosai Dorfman disease (RDD) in 1965. Later Rosai and Dorfman observed as separate entity in 1969 as Sinus histiocytosis with massive lymphadenopathy (SHML). It is a rare self-limiting benign disease. Etiology is unknown, is more prevalent among African Negros and males have more predilection (male: female = 2:1). Although any age group can be affected, most of the patients are younger than 20 years [16]. It presents with gradual painless bilateral cervical lymphadenopathy, fever, raised ESR, hypergammaglobulinemia, and occasionally anemia [17].

FNAC aspirate shows the proliferation of histiocytes with abundant eosinophilic to vacuolated cytoplasm, vesicular nuclei, and lymphophagocytosis or emperipolesis [16]. The diagnosis can be confirmed by using IHC markers. Characteristically, S100 is always positive along with other markers, like CD68, CD163, α1 anti-chymotrypsin, and α1 anti-trypsin, negative for CD1a and Langerin (CD207) [17]. The differential diagnoses include reactive lymph node hyperplasia (RLH), infectious lymphadenitis, Langerhans cell histiocytosis (LCH), non-Hodgkin’s lymphoma, and metastatic carcinoma [18].

2.4 Kimura’s disease

It is a rare chronic inflammatory disorder of unknown etiology. Patients often have enlarged lymph nodes predominantly in the head and neck region [19]. Caused by trauma, allergic reactions to arthropod bites or parasites [20]. It is seen commonly in young adults with the age range of 27–40 years and male to female ratio of 3:1. It is endemic in Asia, especially in China and Japan [19]. Peripheral blood eosinophilia and raised serum IgE levels are usually noted.

FNAC smears shows features of RLH with numerous eosinophils, Warthin-Finkeldey polykaryocytes and epitheloid cell granuloma. IHC cell block for IgE and CD3 may help in cytodiagnosis [21].

2.5 Kikuchi-Fujimoto disease

Kikuchi-Fujimoto disease (KFD) also known as histiocytic necrotizing lymphadenitis is self-limited benign disease. Kikuchi and Fujimoto et al. first described this disease in Asia. It often manifests in young adults (younger than 40 years) [22], but it can occur in any age group with male to female ratio of 1:1 [23]. Most commonly seen in posterior cervical LN, with concomitant involvement of axillary and/or supraclavicular LNs. Infrequently lymphadenopathy is generalized. Most frequent extranodal involvement is skin, bone marrow, and occasionally in the liver. Affected LNs are tender and painful.

Smears examined in such patients show large accumulations of histiocytes and abundant karyorrhectic nuclear debris in a background of necrosis. Neutrophils and eosinophils are rare or absent, an important clue in the diagnosis of this entity. Lysozyme, myeloperoxidase, CD68, CD163, and CD4 are expressed in the histiocytes. Lymphocytes are mostly CD3 positive T-cells. The differential diagnosis includes infectious lymphadenitis, autoimmune lymphadenopathy (primarily SLE lymphadenopathy), and non-Hodgkin lymphoma [22].

2.6 Dermatopathic lymphadenitis

Dermatopathic lymphadenitis (DLN) was first coined by Hurwitt et al. It is also known as lipomelanotic reticulosis [24]. It is a benign form of LN hyperplasia. It is often seen in patients with skin diseases exfoliative or eczematoid inflammatory erythrodermas, neoplastic conditions like mycosis fungoides, Sezary syndrome and uncommonly in the absence of skin disease. Involvement of axillary and inguinal LNs are most commonly seen. Head and neck region LNs involvement is also seen in few patients. DLN is rare entity described in association with human immunodeficiency virus (HIV) infection. FNAC from such patients show numerous noncohesive, pale histiocyte-like cells. Macrophages contain pigment—either hemosiderin or melanin. These have small oval nuclei and a better-defined cytoplasm. Eosinophils may also be seen. The background is predominantly of small lymphocytes which may appear slightly atypical but blast forms are less common [24]. IHC should be considered for confirmation. Histiocytes are S-100, langerin and CD1a positive. DLN is self-limited and often does not require any therapy [25]. Differential diagnosis include Hodgkin’s lymphoma and Mycosis fungoides.

2.7 Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a rare disease, and the estimated annual incidence ranges from 0.5 to 5.4 cases per million persons [26]. LCH may occur at any age, but majority of the cases are seen in children younger than 15 years [26]. It has a varied clinical spectrum from a solitary lesion, to multifocal unisystem to multisystem lesions. The unifocal form usually involves the bone while the multifocal unisystem form always involves the bone. The multifocal multisystem form can involve multiple organs such as bone, skin, liver, spleen, hematopoietic system, and lymph node.

The characteristic cytological features are high cellularity composed of sheets and isolated Langerhans cells (LC) admixed with polymorphous population of cells composed of eosinophils, neutrophils, lymphocytes, plasma cells, and histiocytes. The key to the diagnosis is to identify the LC with its characteristic nuclear features; indentation and nuclear grooves. LCs show positivity for S-100, CD1a, and langerin (CD207). Ultrastructural hallmark is the presence of Birbeck granule [27].

2.8 HIV lymphadenitis

Human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) is a disease caused by the human immunodeficiency virus. LNs are the major sites of emergence and spread of infection. Hence, frequently these patients present with lymphadenopathy. Cytologically it is associated with spectrum of changes from follicular hyperplasia to follicular depletion [28]. Cytological findings are florid follicular hyperplasia in the background of mature lymphocytes, plasma cells, and macrophages. These findings are however not pathognomonic [29]. Presence of many immature cells may be suggestive of lymphoma. Immunophenotyping is helpful in confirming a polyclonal nature of these cells and are mostly B-cells but mature T-cells can also be seen [15, 29].

2.9 Rheumatoid arthritis, systemic lupus erythematosus (SLE)

Smears from patients show numerous small lymphocytes, transformed lymphocytes, and tingible body, some containing Russel bodies [29].

2.10 Castleman’s disease

Castleman’s disease, also known as giant lymph node hyperplasia, is a disease of unknown etiology. Microscopically, based on histological pattern, two types are described: hyaline vascular (HV) and plasma cell type.

Hyaline vascular type is more common. It presents as localized enlarged LN whereas plasma cell type presents with systemic manifestations. FNAC findings in HV type include follicular hyperplasia admixed with lymphocytes, eosinophils, immunoblast, and hyalinised capillaries [30]. Cytological findings in plasma cell type are lymphoid follicular hyperplasia with numerous plasma cells [31].

2.11 Infectious mononucleosis

Infectious mononucleosis (IM), also known as the kissing disease, is a benign, self-limited lymphoproliferative disorder due to exposure to Epstein–Barr virus (EBV) or cytomegalovirus (CMV). The commonest clinical manifestations are pharyngitis, tonsillitis, LN enlargement (typically affecting cervical LNs) [32]. FNAC show lymphohistiocytic aggregates, loose granuloma formations of epithelioid histiocytes and a spectrum of immunoblast maturation. Some of these immunoblast are atypical with large irregular nuclei [33].

2.12 Progressive transformation of germinal center (PTGC)

Benign peripheral lymphadenopathy is often under diagnosed. Etiology and pathogenesis of PTGC is unknown. It can precede or coexist Hodgkin’s lymphoma (HL) or nodular lymphocyte-predominant Hodgkin’s lymphoma (NLPHL). FNAC is characterized by reactive follicular hyperplasia along with mantle zone expansion into the adjacent sinusoids and germinal centers (GC), the follicles with GC become enlarged and are replaced by small lymphocytes [34]. Differential diagnosis are nodal marginal zone B-cell lymphoma, small lymphocytic lymphoma (SLL) and mantle cell lymphoma. Immunostain help in ruling out differential diagnosis. CD3 and CD20 are positive in PTGC in B and T cell regions and are negative for CD15, CD30, and EMA. It is also negative for bcl-2 which is positive in follicular lymphoma [35].

3. Lymphomas

Lymphomas are neoplastic proliferations of lymphoid origin. Broadly classified as Hodgkin’s and Non-Hodgkin’s lymphomas, they can be both picked up on FNAC which is also the first line of investigation in their workup. Role of FNAC here is to diagnose, subclassify and stage the lymphomas. World Health Organization’s (WHO) classification [36] is relatively easy to apply to cytology smears and requires detailed cytomorphological, immunophenotypic and molecular features which are easily obtained by using ancillary techniques on cytology material.

3.1 Hodgkin’s Lymphoma

The global incidence of Hodgkin’s lymphoma is 0.98 per 100,000 population [37]. It shows bimodal peak with first peak in young adults of 15–30 years and second peak in old age [7, 12]. A third peak is believed to occur in childhood in developing countries. About 70% patients present with lymphadenopathy affecting the cervical group of lymph nodes, followed by mediastinal, axillary, and paraaortic nodes [5, 12]. The systemic manifestations including fever, night sweats, and significant weight loss may be seen in advanced disease. The WHO has classified Hodgkin lymphoma in following categories [36].

1. Classical Hodgkin lymphoma (CHL)

   • Nodular sclerosis

   • Lymphocyte rich

   • Mixed cellularity

   • Lymphocyte depletion

2. Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)

3.1.1 Classical Hodgkin lymphoma

The smears show the presence of Reed-Sternberg (R-S) cells lying in a polymorphic cell population comprising of small mature lymphocytes with eosinophils, plasma cells, histiocytes, and immunoblasts in varying proportions. The classical R-S cells are large in size ranging from 20 to 50 μ in diameter, have moderate to abundant amphophilic cytoplasm with usually bilobed nuclei however true binucleation or multinucleation may also be seen. The nuclei have coarsely clumped chromatin and two centrally placed prominent nucleoli which appear pale gray with MGG stain [6, 7, 12]. A perinuclear clear halo is also frequently seen. The classical R-S cells are easily demonstratable in mixed cellularity HL however this typical morphology is often lacking in other subtypes of CHL. The variants include mononuclear cells which contain single nucleus with all the above-described features (Hodgkin cells). The lacunar type of R-S cell is characteristic of nodular sclerosis Hodgkin lymphoma in histological sections however is occasionally found in cytology smears. These are large cells having abundant pale cytoplasm with indented or overlapping segmented nuclei. The nodular sclerosis HL is the most common subtype, yield scanty aspirate with an increased number of fibroblasts and collagen material. The hypocellular smears often result in false negative interpretations. The R-S and Hodgkin cells in mixed cellularity HL are present in a background comprising of small- to medium-sized lymphocytes, eosinophils, plasma cells, centroblasts, and histiocytes with an epithelioid morphology. This subtype may mimic suppurative lymphadenitis in cases where neutrophils dominate. The lymphocyte-rich HL shows rare R-S cells and Hodgkin cells of the above-described features but a background rich in lymphoid cells exhibiting full spectrum from mature lymphocytes to germinal center formation. Eosinophils and histiocytes are usually not seen in this subtype.

3.1.2 Nodular lymphocyte predominant Hodgkin lymphoma

The smears of NLPHL show the presence of L and H cells in a background chiefly composed of small mature lymphocytes. These are giant cells with scant cytoplasm and multilobated extremely folded popcorn-like nucleus with vesicular nuclear chromatin and many small nucleoli. The diagnosis of NLPHL on FNA smears is challenging as L and H cells are usually difficult to find and are outnumbered by a sea of reactive small B and T lymphocytes.

Cytologically, NLPHL may be confused with T-cell-rich large B-cell lymphoma which shows abundant reactive small lymphocytes with only a few neoplastic cells [10]. These cells on IHC show uniform positivity for CD20 and are negative for CD15 and CD30, thus ruling out the possibility of CHL.

Anaplastic large cell lymphoma (ALCL) also shows large multinucleated cells which may be indistinguishable from R-S cells though neoplastic cells in ALCL are much more numerous in contrast to HL, wherein the R-S cells comprise only about 0.1–10% of the total cell population [10]. In addition, ALK1 positivity and gene rearrangement studies are helpful in the diagnosis of former [15].

Nasopharyngeal carcinoma commonly metastasizes to cervical LNs and shows large neoplastic cells simulating R-S cells in an abundant lymphoid background on cytology. Immunostaining with CK and EMA may be used to exclude HL in such cases.

A variety of other benign conditions like infectious mononucleosis, toxoplasmosis, drug-induced lymphadenopathy, post vaccinal lymphadenopathy may show large atypical immunoblasts with similar morphology as R-S cells, however these cells usually have coarse nuclear chromatin and smaller nucleoli.

The Hodgkin and R-S cells of CHL show positive immunostaining with both CD15 and CD30 and are negative for CD45, EMA and usually negative for both B and T-cell markers. Those associated with EBV are positive for EBV-LMP [5, 7, 12].

The L and H cells of NLPHL are positive for CD45 and CD20 and are positive for EMA in 50% cases. They are however negative for CD15 and may show occasional positivity for CD30 [5, 7, 15].

There is limited role of FCM in diagnosis of HL. An increase in the CD4+ T cells with a CD4+/CD8+ of more than 4:1 may serve as a clue, however, is not diagnostic. NLPHL may show an increased proportion of CD4 + CD8+ (double positive) or CD57+ T-cells [6, 10].

3.2 Non-Hodgkin lymphoma

3.2.1 Small cell lymphoma

Small cell lymphomas are a group of lymphomas, with predominate monotonous cell type on first inspection of cells (Table 3). They are encountered on regular basis by cytopathologist and needs a good understanding of various differentials to make correct diagnosis highlighted in Table 4.

Precursor B lymphoid neoplasms	Precursor T lymphoid neoplasms
B-lymphoblastic leukemia/lymphoma, NOS	T-lymphoblastic leukemia/lymphoma
B-Lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities
Mature B-cell neoplasms	Mature T-cell neoplasms
B-cell prolymphocytic leukemia	T-cell prolymphocytic leukemia
Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma	T-cell large granular lymphocytic (LGL) leukemia
Follicular lymphoma	NK-lymphoblastic leukemia/lymphoma
Mantle cell lymphoma	Adult T-cell leukemia/lymphoma (ATCL)
Marginal zone lymphoma—Nodal and extranodal	Angioimmunoblastic T-cell lymphoma (AITL)
Lymphoplasmacytic lymphoma	Anaplastic large cell lymphoma, (ALCL) T/null-cell
T-cell/histiocyte-rich large B-cell lymphoma	Mycosis fungoides
Burkitt lymphoma	Hepatosplenic T cell lymphoma
Diffuse large B-cell lymphoma (DLBCL), NOS

Table 3.

Simplified WHO classification for Non-Hodgkin’s lymphoma.

Differential diagnosis	Cytology	Immunocytochemistry
Mantle cell lymphoma	Monomorphic population of small lymphoid cells	Positive: CD5, FMC-7, Cyclin D1 Negative:CD10, CD23, CD38
Small cell lymphocytic lymphoma	Admixed larger paraimmunoblasts and centroblasts	Positive: CD23, CD5 Negative:CD10, CD38, FMC-7, Cyclin D1
Follicular lymphoma		Positive: CD10, BCL2, CD23 Negative: CD5, CD38, Cyclin D1
Marginal zone lymphoma	Heterogeneous population of cells including monocytoid cells, plasma cells, and centroblasts	Positive: Surface Ig Negative: CD5, CD10, CD23, CD38, Cyclin D1
Lymphoplasmacytic lymphoma	Small lymphocytes, plasmacytoid, and plasma cells. PAS positive intranuclear inclusions	Positive: CD38 Negative: CD5, CD10, CD23, Cyclin D1

Table 4.

Types of small cell lymphomas and their distinguishing features [7].

Next, individual small B cell NHLs are discussed in detail, to provide all the available information to the cytopathologists and help them in differentiating these similar pathologies.

3.2.1.1 Mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma, which mainly affects elderly men. MCL represents 3–10% of all NHLs. Most common presentation is generalized lymphadenopathy and hepatosplenomegaly, with lymphoma cells seen in blood of 25% of cases. Sometimes extranodal sites like gastrointestinal tract are involved. The median survival is 3–5 years.

Cytology smear shows a monomorphic population of small to intermediate cells with nuclear membrane irregularities and indentations, fine nuclear chromatin, inconspicuous nucleoli, and scant cytoplasm. An aggressive blastoid variant can have intermediate to large cells with large nuclei having irregular margin, finely dispersed chromatin, and multiple small nucleoli. Blastoid variant has high mitotic activity [7, 38, 39].

Neoplastic cells express CD19, CD20, CD5, CD79b, Cyclin D1, and FMC7 while negative for CD10 and CD23. Light chain restriction is seen mostly in the form of kappa restriction.

Mantle cell lymphoma shows diploid cells while blastoid variant mostly shows a tetraploid population with high proliferative activity, suggestive of a high-grade lymphoma [39].

Characteristic genetic abnormality seen is t(11;14) (q13q32) translocation, leading to overexpression of cyclin D1, a cell-cycle protein [40].

3.2.1.2 Follicular lymphoma

Follicular lymphoma is a tumor of follicular center B-cells. It is a common NHL, constituting 20–35% and is seen mostly in elderly patients. Most common presentation is generalized lymphadenopathy with spleen and bone marrow involvement [7, 36]. This is a mildly aggressive lymphoma with high 5 years survival rate.

Follicular lymphomas show a mixture of small, irregular lymphocytes, and larger cells. The small lymphocytes, have irregular nuclear contours and inconspicuous nucleoli while larger cells (centroblasts) are characterized by sharply demarcated basophilic cytoplasm, round, noncleaved nuclei with finely granular chromatin and prominent 2–3 peripheral nucleoli.

FL is positive for CD19, CD20, CD10, and bcl-2 while it is negative for CD5.

Neoplastic cells show t(14;18)(q32; 21) translocation that results in the rearrangement of the bcl-2 gene and in the expression of the “antiapoptosis” gene [41].

3.2.1.3 Lymphoplasmacytic lymphoma

Lymphoplasmacytic lymphoma (LPL) accounts for only 1.5% of all nodal lymphoma and is seen mostly in elderly. Most common involvement is seen in lymph node, spleen, and bone marrow along with peripheral blood. Elevated levels of monoclonal serum paraprotein of the immunoglobulin M type are seen in most of the patients that sometimes result in symptoms of hyperviscosity (Waldenstrom macroglobulinemia). LPL shows an indolent course and may very rarely transform into DLBCL.

Cytology smears show abundant small lymphocytes, plasmacytoid, and plasma cells. Occasionally, PAS-positive intranuclear inclusions known as Dutcher bodies may be observed.

Neoplastic cells are positive for CD19, CD20, CD22, CD79a, CD38 and shows light chain restriction while being negative for CD5, CD10, CD23.

Neoplastic cells may show a variety of immunoglobulin heavy chain and light chain genes rearrangement, but no single characteristic chromosomal abnormality is known.

3.2.1.4 Marginal zone lymphoma

Marginal zone lymphoma (MZL) is a relatively uncommon low-grade B-cell lymphoma. MZL may be nodal or extranodal. Extranodal MZL is commonly known as MALT lymphoma and constitute 7–8% of all lymphomas. Most common sites of extranodal MZL are stomach, eye, thyroid, salivary gland, lung, and skin [42]. MALT lymphoma shows a strong association with autoimmune diseases, such as Hashimoto’s thyroiditis, Sjogren’s syndrome, and Helicobacter pylori infection-induced gastritis.

Cytology smear shows a heterogeneous population of monocytoid cells, small, cleaved cells, large cells, and plasma cells. The monocytoid cells have moderate to abundant amounts of pale cytoplasm. The nuclei can vary from oval to reniform shape, with vesicular or coarse chromatin and inconspicuous nucleoli [43].

Neoplastic cells are positive for CD19, CD20, CD22 and surface Ig while are negative for CD5, CD10, CD23, and CD103.

Trisomy 3 and t(11;18), (q21;q21) translocations have been reported in extranodal MZL [44].

3.2.1.5 Small lymphocytic lymphoma

Small lymphocytic lymphomas (SLL) are seen mostly in older adults, with bone marrow and peripheral blood involvement in many cases. It is an indolent disease. It may undergo transformation into high grade lymphoma like prolymphocytic lymphoma (PLL) or large-cell lymphoma (Richter syndrome).

SLL shows a monomorphic population of small, round lymphocytes with nuclei that have a checkerboard pattern of clumped chromatin. Prolymphocytes and paraimmunoblasts are scattered in the background. Prolymphocytes are slightly larger than paraimmunoblasts, but both have gray-blue cytoplasm, round nuclei and a prominent nucleolus (Figure 2). Unfavorable cytological features are, increase in the number of paraimmunoblasts, presence of plasmacytoid cells, mitotic figures, apoptotic bodies, necrosis, and a myxoid, dirty background [45].

Figure 2.

Small lymphocytic lymphoma showing mature lymphocytes with round nucleus, coarsely clumped chromatin and indistinct nucleoli (MGG-600×).

Neoplastic cells are positive for CD19, CD20, CD5,CD43, CD23 and shows light chain restriction while they are negative for CD10 and FMC7.

Deletion of chromosome 13q14 is the most common genetic abnormality, followed by trisomy 12 [46].

3.2.1.6 Burkitt lymphoma

Burkitt lymphoma (BL) is a highly aggressive and potentially curable lymphoma commonly occurring in children. It can occur in three clinical settings. (1) The endemic BL is seen almost exclusively in children aged 4–7 years old in Africa and to a lesser extent in Middle east and south America. Jaw and facial bones are the most common sites, other extranodal sites being intestine, breast, and ovary. (2) The sporadic variant commonly presents as an abdominal mass and occurs in children and young adults. (3) The immune deficiency-associated variant is commonly seen to be associated with HIV. BL has a very high tumor burden with a doubling time of 24 hours in some cases [47]. EBV is seen to be commonly associated with BL: almost all cases of endemic, 30% cases of sporadic and 25–40% cases of immune deficiency associated BL [48].

The aspirates are hypercellular with a uniform monotonous population of medium-sized cells with round nuclei, coarse chromatin, and multiple (2–5) small but prominent nucleoli. The cytoplasm is abundant, blue, and vacuolated (especially in air dried Romanowsky smears) (Figure 3). The formalin-fixed cell blocks as well as tissue sections have a starry sky appearance due to the abundance of tingible body macrophages and numerous apoptotic cells (Figure 4).

Figure 3.

Large cell lymphoma (Burkitt lymphoma) dirty proteinaceous background with cells showing marked pleomorphism, scanty to moderate cytoplasm, indented nuclei and frequent mitosis (MGG; 400×).

Figure 4.

Large cell lymphoma (Burkitt lymphoma) dirty proteinaceous background with cells showing marked pleomorphism, scanty to moderate cytoplasm, indented nuclei and frequent mitosis (Hematoxylin and eosin 200× cell block preparation).

The diagnosis of BL is imperative because it warrants a more aggressive chemotherapy regimen than DLBCL. The cells of BL are positive for CD19, CD20, CD10, sIg, IgG heavy chain or kappa/lambda light chains and BCL6. Ki67 index is usually 100%. The diagnosis is done based on morphology, characteristic immunophenotype, and demonstration of MYC translocation via karyotyping or FISH. Demonstration of MYC translocation can also be done by ICC/IHC, with nuclear immunoreactivity in >40% of cells termed as positive [49]. The most common translocation (80%) is t(8,14) where MYC translocate from chromosome 8 to Ig heavy chain on 14. Other translocations seen are t(2,8) and t(8,22) where the translocation occurs with the light chain. MYC translocation is highly characteristic but not specific.

3.2.2 Large cell lymphomas

3.2.2.1 Diffuse large B cell lymphoma

Diffuse large B cell lymphoma is a very commonly encountered NHL and comprises 30–40% of adult NHLs. Extranodal disease is seen in about 40% of patients, with stomach being the most common site [50]. Previously, three morphologic subtypes were recognized which later proved to be clinically irrelevant. Two molecular subtypes are now recognized based on gene expression profiling: germinal center B cell (GCB) like and activated B cell (ABC) like with the latter having poor prognosis and poor survival after chemotherapy.

FNA smears show variable cellularity with mediastinal and extranodal sites frequently having low cellularity. This might be because of extensive sclerosis or sparse distribution of cells commonly seen in these sites. However, most smears are moderately cellular with sheets of large, atypical cells. The cells are categorized as large when the nucleus size is more than histiocytes nucleus or twice more than a lymphocyte’s nucleus.

The centroblastic variant comprises of a predominant population of centroblasts with characteristic round nuclei and multiple small nucleoli. The immunoblastic variant has a pleomorphic cell population with large blast cells having abundant blue cytoplasm, perinuclear pale zone and round-irregular nucleus with single prominent nucleoli. Anaplastic variant has cells with bizarre pleomorphic nucleus with multinucleation. These cells may resemble RS cells or ALCL cells (Figure 5).

Figure 5.

Diffuse large B-cell lymphoma-highly cellular smear with large atypical lymphoid cells, marked nuclear pleomorphism and high N:C ratio (MGG; 1000×).

Cell blocks are useful to differentiate these large cell lymphomas from indolent low-grade ones by providing architectural insight. DLBCL is positive for CD19, CD20 and CD10 (Figure 6). Approximately 40–60% of these lymphomas are positive for MUM1 and BCL6 is positive in majority (60–90%) [51]. Aberrant co-expression of CD5 and aberrant loss of immunoglobulin expression can also be seen. The GCB type DLBCL is positive for BCL6, negative for MUM1, and positive/negative for CD10. The non-GCB subtype (including ABC) are either double negative for CD10 and BCL6 or positive for both BCL6 and MUM1. Positivity for both BCL2 and MYC on IHC classifies it as double expressor DLBCL, seen in about 30% of DLBCL, NOS cases [52]. Positivity is defined as >30% cells expressing the marker. These cases portend a poor prognosis for the patient with relapse and CNS involvement being more commonly seen. If MYC and BCL2 rearrangements are confirmed by FISH, it used to be termed double hit DLBCL, but it has been categorized as a separate entity now: high grade lymphoma with MYC and BCL2 rearrangement. Virtually all cases of DLBCL have heavy or light chain gene rearrangements. Some cases might have t(14,18), if there is a history of follicular lymphoma. On FCM, the large cells may appear outside of the usual lymphoid gate with higher SS and FS.(Sometimes, the cell count in FCM is low as the large cells are fragile and may not survive processing. However in most of the cases on gating CD45 dim population with CD10 (mayor may not be present) CD19 and CD79 expression with light chain expression (Figure 7).

Figure 6.

Diffuse large B-cell lymphoma-CD20 positive large atypical lymphoid cells (ICC; 600×).

Figure 7.

Expression of CD19 and kappa chain restriction (FCM).

3.2.2.2 T cell/histiocyte rich B-cell lymphoma

It is subtype of DLBCL where histiocytes and reactive T cells are present in such abundance as to obscure the tumor cells. It commonly affects men in middle age [53]. The smears are cellular with a predominant population of small mature lymphocytes. Variable number of scattered histiocytes are also seen along with scattered large cells such as anaplastic, centroblasts, popcorn like or occasionally RS cells. It is diagnosed on cytological smears with difficulty and usually immunophenotyping on cell/tissue blocks is needed for definitive diagnosis. The smaller cells are positive for CD3, CD5, and CD8 and the large cells express DLBCL phenotype.

3.2.2.3 Precursor lymphoblastic lymphoma/leukemia

Precursor Lymphoblastic Lymphoma/Leukemia (LBL) is commonly seen in older children or young adults and is a neoplasm of immature lymphoid ells. T-LBL is more common and constitutes 90% of lymphoblastic lymphomas [54]. The patients usually present with a mediastinal mass and can rapidly progress to involve the peripheral blood, bone marrow, CNS, and gonads. B-LBL is less common and involves lymph nodes, skin, and even bones.

The FNA smears are hypercellular and show a homogenous population of small to medium-sized cells with high N:C ratio, lobulated, convoluted, or round nuclei and speckled chromatin with 2–3 prominent nucleoli. There will be frequent mitosis and B and T LBLs are morphologically similar. Certain clues like exaggerated convolution, cerebriform nuclei point toward T-LBL.

Demonstration of immunoexpression of TdT by IHC or FCM that occurs exclusively in LBL help in differentiating them from rest of the lymphomas. T-LBL would demonstrate positivity with surface/cytoplasmic CD3 and other T cell markers like CD5, CD7 depending on the stage of development. Other markers include CD2, CD34, CD4, and CD8. 20% of T-LBLs show positivity for NK cell markers like CD16 and CD56 [55].

B-LBL expresses B lineage markers like CD19, CD79a, and PAX5. CD20 is positive in 50% of the cases. CD10 is positive in the CALLA variant. While surface IG are usually negative, cytoplasmic Ig can be expressed in pre-B LBLs.

3.2.2.4 Anaplastic large cell lymphoma (ALCL)

Anaplastic large cell lymphoma (ALCL) is a separate entity for T-cell and null cell types in WHO classification of haematolymphoid neoplasms. It accounts for about 3% of adult non-Hodgkin lymphomas and 10–20% of childhood lymphomas [56]. ALCL affects children and young adults but can occur in any ages. Apart from lymph nodes, the extranodal sites involved are skin, bone, lung and soft tissues.

The aspirate smears are moderately to highly cellular. The most common morphologic variant of ALCL is pleomorphic type in which large pleomorphic cells with eccentric, horseshoe-shaped or kidney-shaped nuclei are present: hallmark cells. These cells have fine or clumped chromatin with prominent basophilic nucleoli and usually abundant amphophilic cytoplasm. Another characteristic cell type are Doughnut cells which are multinucleated giant cells with abundant clear or basophilic cytoplasm and multilobate nuclei in wreath like pattern. There is a paucity of lymphoglandular bodies. Necrosis and inflammation are also seen commonly. Small cell variant of ALCL has small to medium sized cell with irregular nuclei and moderate amount of cytoplasm. The other two common morphologic types are small cell variant (that might resemble PTCL) and lymphohistiocytic variant which has numerous histiocytes that might mask the tumor cells. Both small cell and lymphohistiocytic variant have a worse prognosis with more tendency for leukemic spread [57].

IHC on cell block with CD30 markers shows perinuclear dot-like or strong membranous positivity while being negative for CD15. ALCL also expresses T-cell markers such as CD2 and CD4 but 15% cases have null-type phenotype [58]. The chromosomal translocation t(2,5) is seen where ALK gene on chromosome 2 fuses with NPM (nucleophosmin) gene on chromosome 5. This gene product (ALK) can also be demonstrated by ICC. EMA immunoexpression also helps to differentiate ALCL from other lymphomas. FCM is rarely useful with necrosis and surrounding inflammation confounding the results. Demonstration of TCR-gamma/delta is usually helpful to establish clonality. FISH can also be used for demonstration of the translocation using the break apart probe.

3.2.2.5 Peripheral T-cell lymphoma

Peripheral T-cell lymphoma (PTCL), has a mature or post thymic phenotype, unspecified accounts approximately half of the case of PTCL. PTCL affects adult population more commonly but can also affect children. Along with generalized lymphadenopathy, there might be involvement of bone marrow, liver, spleen, and extranodal tissues too.

The aspirate smears of PTCL are highly cellular and pleomorphic. They show a spectrum of atypical cells from large to small cell type [59]. The larger cells have large single to multiple nuclei that have fine to clumped chromatin, smooth to irregular border and huge nucleoli (Figure 8). The presence of multiple nuclei and prominent nucleoli may give the appearance of RS cells/variants. However, cytoplasm is usually abundant and pale gray and may point toward it being a T-cell lymphoma rather than B-cell. The small morphologic type has tumor cells which are only slightly bigger than small mature lymphocytes, however have irregular nuclear contour with nuclear indentations, cleaving and folding. The nuclei have clumped chromatin and inconspicuous nucleoli. Intermediate sized cells of PTCL show features which are a mix of both large and small cell type.

Figure 8.

T-cell lymphoma; large pleomorphic cells with indented, folded nuclei and 1–2 distinct nucleoli (MGG; 1000×).

ICC can be used to demonstrate the T cell phenotype. There is variable expression of pan T-cell markers like CD3, CD5, and CD2. CD7 expression is seen to be frequently lost. There is variable expression of CD4 and CD8 and CD4 positivity is more commonly seen than CD8 (Figure 9). The cells can also be dual negative or dual positive for CD4 and CD8. CD30 and CD52 are other markers that can be positive in 50% of the tumor cells [60, 61]. FCM can be helpful to isolate the clonal subset with aberrant T-cell phenotype but usually falls short of making a definitive diagnosis. PCR can be used to demonstrate T-cell receptor rearrangement.

Figure 9.

Expression of CD7 and CD34 (on FCM).

4. Metastatic lymph node disease

Lymph nodes contain blood, lymphatic vessels and stromal tissue.

Metastatic disease of LN often shows diffuse involvement and can be picked up on cytology. FNAC is of considerable value in disease staging and documentation of metastasis in both known and occult tumors. In more than 90% cases of lymph node metastasis, primary site of origin is already known [62].

Carcinomas show lymphatic pattern of dissemination thus lymph nodes are commonly involved. In well-differentiated squamous cell carcinoma (SCC), the tumor cells have hyperchromatic nuclei with individual cell keratinization. The tumor cells often show necrotic background. Thus, in case of low cellularity smears with abundant necrosis, a careful search for the tumor cells is required (Figure 10) [63]. Branchial cleft cyst and epidermal inclusion cyst are close differentials due to presence of amorphous debris with necrotic parakeratotic cells. Poorly differentiated squamous cell carcinoma which might demonstrate cell clusters showing thick nuclear membrane and prominent nucleoli pose a diagnostic challenge and often becomes difficult to distinguish from adenocarcinoma [64].

Figure 10.

Squamous cell carcinoma cellular smears with necrotic background showing polygonal cells with intercellular bridges, blue cytoplasm, Ink dot pyknotic nuclei and bizzare nuclear forms. Also seen are tumor giant cells (MGG; 400×).

In Metastatic adenocarcinoma, the cells are usually arranged in acinar or glandular pattern and are large, cuboidal to columnar with moderate amount of cytoplasm and pleomorphic nuclei with prominent nucleoli. Vacuolated cytoplasm usually indicates intracellular mucin accumulation (Figure 11). Presence of intracellular mucin and necrosis, usually suggests gastro intestinal tract as primary site of origin. Moderately pleomorphic glandular cells with gland-in gland formation suggests metastasis from prostatic carcinoma. Metastasis from renal cell carcinoma shows large cells with abundant pale, granular vacuolated cytoplasm and prominent nucleoli. Similarly, large cells with prominent nucleoli supports the diagnosis of anaplastic carcinoma of lung and nasopharynx. Metastatic deposit of Papillary thyroid carcinoma displays a papillary pattern with central fibrovascular core along with nuclear grooving and intranuclear inclusion. Metastatic ductal carcinoma of breast shows high cellularity with several loose clusters of tumor cells having moderate to abundant cytoplasm with pleomorphic nuclei and prominent single to multiple nucleoli [63].

Figure 11.

Cuboidal to columnar cells in acini with palisaded glands. Cells show cytoplasmic extension, vacuolation, moderate nuclear pleomorphism, and compact chromatin (MGG; 40×).

The role of FNA for searching unknown primary tumor can be facilitated by ICC on aspirated smears/cell block. In metastasis from epithelial tumors, the IHC/ICC expression of CK7 and CK20 profile helps to triage according to the origin from primary tumor. Table 5 summarizes CK7 and CK 20 immunohistochemical markers in various epithelial malignancies.

CK7+/CK20−	CK7−/CK20+	CK7+/CK20+	CK7−/CK20−
Breast carcinoma; Lung adenocarcinoma; Endometrial adenocarcinoma; Endocervical adenocarcinoma; Ovarian (serous) carcinoma; Small cell lung carcinoma; Thyroid carcinoma; Salivary gland tumors; Kidney (papillary); Gastric adenocarcinoma	Colorectal adenocarcinoma; Merkel cell carcinoma	Urothelial carcinoma; Pancreatic adenocarcinoma; Bladder carcinoma; Cholangiocarcinoma	Prostatic adenocarcinoma; Renal (clear cells); Hepatocellular carcinoma; Adrenocortical carcinoma

Table 5.

Immunohistochemistry for carcinoma of unknown primary.

Though the risk of LN metastasis is low in malignant round cell tumors (SRCT), they may be seen on LN aspirates, for example, Ewing sarcoma, rhabdomyosarcoma, retinoblastoma, neuroblastoma, hepatoblastoma, Wilms tumor and osteogenic sarcoma (small cell variant). These tumors can be differentiated with the help of IHC markers. Cytologically, these tumors are composed of uniform population of round to oval cells with scanty, basophilic cytoplasm; mixed populations of small and large cells present in some tumors like neuroblastomas. These tumors consist of cells size of approximately three times the small mature lymphocyte and hyperchromatic nucleus with evenly distributed chromatin with cytoplasm usually scanty with lack of nucleoli and very high nucleo-cytoplasmic ratio [63]. Metastasis from small cell carcinoma undifferentiated type arising from lung show cohesive clusters of tumor cells with scant cytoplasm, coarse chromatin, frequent mitosis and necrosis thus, it is important to differentiate from small and medium sized lymphoma and large cell lymphoma [65].

Metastasis from malignant melanoma have dissociated cells, well defined cytoplasm, eccentric nuclei, dense chromatin, and rarely cells have intracytoplasmic pigment. IHC analysis for S-100, Melan A and HMB45 are of great value in diagnosing the melanoma [66].

Metastatic sarcomas are very uncommon in lymph nodes [67]. Cytological features of sarcomas are varied in different types. More common sarcomas metastatic to lymph node include synovial sarcoma, Kaposi sarcoma, Follicular dendritic cell sarcoma, epitheloid sarcoma rhabdomyosarcoma. and epithelioid sarcoma.

5. Lymph node FNA cytology reporting using Sydney system

The current WHO classification of lymphoproliferative disorders incorporates clinical, morphological, and ancillary data that are required for specific diagnoses. Despite the tremendous progress made in performing and interpreting LN-FNAC and its correlation with ancillary tests, the technique is not uniformly accepted by clinicians and pathologists. This is mainly due to the lack of widely shared and accepted guidelines and a cytopathological classification that directly relates to management. The proposal of the Sydney system for performing classification and reporting of lymph node cytopathology was published by an expert panel. Table 6 highlights the proposed Sydney classification for reporting the lymph node cytopathology [68].

1.Inadequate/non-diagnostic	Blood only; Necrosis	Repeat FNAC
2. Benign	Acute lymphadenitis; Reactive hyperplasia; Granulomatous lymphadenitis; Necrotising granulomatous lymphadenitis	1.PCR and microbiological culture for organisms.; 2.Methanamine staining of a cell block to confirm or demonstrate a specific fungal infection.; 3.Flow cytometry confirming a reactive lymphoid population.
3. Atypical cells of undetermined significance/atypical lymphoid cells of uncertain significance.	Atypical non-lymphoid cell; Atypical lymphoid cell	1.Flowcytometery; 2.Core needle biopsy; 3.Wait and watch.
4. Suspicious for malignancy	Suspicious for metastasis; Suspicious for lymphoid malignancy	1.Flowcytometery; 2.Cell Block IHC; 3.If no ancillary test available: Excisional biopsy.
5. Malignant	Metastases; Metastatic squamous cell carcinoma Metastatic adenocarcinoma; Metastatic breast carcinoma; Metastatic small cell carcinoma; Metastatic melanoma; Metastatic poorly differentiated carcinoma; Metastatic thyroid carcinoma; Non-Hodgkin’s lymphoma; Hodgkin’s lymphoma; Leukemia infiltration; Burkitt lymphoma	1.IHC on a cell block to diagnose the type and origin of a metastatic carcinoma.; 2.Flow cytometry immunophenotyping and conventional cytogenetics or FISH for specific type of lymphoma.

Table 6.

Sydney system classification.

Fine needle aspiration cytology is an invaluable tool in the workup of lymphadenopathy, neoplastic and non-neoplastic. Lymph nodes offers the most fulfilling diagnostic experience to a cytopathologist. This is considered first line investigation for superficial or deep seated and inoperable lymphoid masses.

The introduction of a standardized reporting system viz The Sydney system shall help in streamlining the quality of procedure, optimal utilization of material for ancillary testing and better understanding of the report communicated to the clinician, overall aiding patient care. With proper utilization of FNAC, unnecessary surgical interventions in cases of infective, lymphoproliferative, and metastatic disorders might be avoided. The ever increasing use of FCM and molecular testing in the diagnostic workup of lymphoproliferative disorders and the ease and suitability of FNAC in obtaining tissue sample for these testing, an increasing reliance on FNAC as preferred modality can be foreseen.