Aspiration biopsies of lymph nodes should preferably be performed with a 23 gauge (0.6 mm) needle. In most cases this will provide enough cells for smears as well as cytospin preparations. The use of larger needles usually results in admixture of peripheral blood which may preclude cytological and immunological evaluation of the lymphoid cells.

Algorithm Lymphadenopathy clinical management.

Lymph node aspirates are usually cellular, making it difficult to prepare smears of good quality. The smear should be thin to ensure instant fixation, which will allow an optimal evaluation of cytological details. However, care must be exercised not to use too much pressure in preparing such thin smears. Lymphoid cells are fragile and readily lose their cytoplasm. Fragmented cytoplasm will appear as small pale grey structures with Romanowsky stains, and these are often called ‘lymphoglandular bodies’. Sometimes identical structures can also be seen in smears from other fragile cells; they are thus not pathognomonic for cells of lymphoid origin.

Whenever possible, both air-dried and alcohol-fixed smears should be prepared for May–Grünwald–Giemsa (MGG) and Papanicolaou (PAP) staining, respectively. These stains complement each other and allow an optimal evaluation of cytological details. If mycobacterial or fungal infections are suspected, extra material should be sent for PCR and/bacteriology.

After using parts of the aspirates for smear making, the remainder should be suspended in a buffered balanced salt (BBS) solution at pH 7.4 for cytospin preparations. An ordinary aspirate from an enlarged lymph node will yield several millions of cells. The number of suspended cells should therefore be calculated and the concentration adjusted to 1–2 × 10⁶ cells/ml. Cell-rich suspensions can be diluted to optimal concentration by adding BBS solution. Vigorous mixing should be avoided since it can destroy lymphoid cells, particularly the large immature cells seen in high-grade lymphomas. If the cell concentration is low the cells can be concentrated by centrifugation at 700 rpm for 3–5 min. The resulting pellet is then gently resuspended in a reduced volume of BBS solution. To prepare the cytospin slides the cell suspension is spun in a cytocentrifuge at 700 rpm for 3 min. Each cytospin should contain 1–2 × 10⁵ nucleated cells.

One of the cytospins should always be stained with MGG and compared with the smears to monitor recovery of all cell components. If the suspension contains a rich admixture of red blood cells, it is possible to purify the lymphoid cells by density gradient centrifugation. Normally this procedure does not result in any significant cell loss except in some cases of large cell lymphomas which may be fragile and therefore lost in density gradient centrifugation.

Air dried cytospin preparations can be stored at room temperature for up to 1 week without detrimental effect on the immunological staining. Alternatively the cytospin can be stored at −20°C either in a plastic box or wrapped in aluminium foil. Under these conditions lymphoid cells retain their immunological and morphological characteristics for at least 1–2 years. It is important that the slides are kept wrapped until fully thawed when brought out for use otherwise the cells are prone to disintegration. Both immunoalkaline phosphatase and immunoperoxidase methods are suitable for cytospin preparations.

Aspirated cells can also be immunologically characterised by flow cytometry (FC).^{14,15,18–20,23,26} As in the case of immunocytochemistry on cytospin preparations one part of the aspirate should be used for smear making. The second part should be suspended in BBS solution at pH 7.4. A cell concentration of approximately 1 million cells per ml buffer will be sufficient for a complete characterisation of reactive lesions as well as most B- and T-cell lymphomas. At the moment four colour FC is standard in immunophenotyping of lymphomas. Evaluation of scattering light allows elimination of dead cells and granulocytes. Several studies have shown a good agreement between FC on FNA material and surgical biopsy specimen.^21,25

FC is a rapid and sensitive technique which can detect small abnormal cell populations in a reactive background. Since FC does not allow an evaluation of cytomorphology it is of importance that the results are correlated to cytomorphology on routinely stained smears. Lymphomas with large cells are often fragile and such cells are destroyed during FC analysis. A close cooperation between the FC laboratory and the cytopathologist is therefore strongly recommended.

Aspirated cells also perform well in PCR rearrangement analysis.^27,28,30 After the aspirated cells have been suspended in BBS at pH 7.4 they should be pelleted immediately, snap frozen and stored at −70°C until used for rearrangement analysis. Cytospin preparations can also be used for FISH analysis of specific translocations to aid subtyping of lymphomas.

The lymph node parenchyma is surrounded and divided by a fibrous capsule with attached septa. The parenchyma is composed of the cortex, medulla and paracortex. B cells predominate in the cortex and medulla whereas T cells are mainly found in the paracortical tissues.

The cortex contains primary and secondary follicles, the proportions varying with the state of activity of the node. Primary follicles, composed of aggregates of small resting B cells, are found in the unstimulated node. Secondary follicles develop after antigen stimulation and are composed of a narrow mantle zone of small B lymphocytes surrounding a germinal centre. Several types of cells are found in the germinal centre, the vast majority being B cells in the form of centroblasts and centrocytes. Macrophages containing phagocytosed cellular debris are also present.

Mature immunoglobulin secreting B cells, familiar as plasma cells, are the principal cell type found in the medulla. The paracortex contains many small lymphoid cells which are of T phenotype. In addition activated T cells and immunoblasts are present.

Normal cytology (Fig. 13.1)

As could be predicted from the description of the normal histology, aspirates from normal lymph nodes and from some reactive nodes are dominated by different types of lymphocytes, but plasma cells, macrophages and granulocytes are also found.

Fig. 13.1 Lymph node aspirates with normal lymphoid cells (A). A macrophage with plentiful clear cytoplasm containing ‘tingible bodies’ is surrounded by large round centroblasts with sparse cytoplasm. Medium-sized centrocytes with irregular nuclei and smooth chromatin and small mature lymphocytes with condensed chromatin are also present (MGG). (B). One large immunoblast with an eccentrically placed nucleus and well developed basophilic cytoplasm is surrounded by smaller centroblasts and mature plasma cells (MGG).

Histologically, this response may take the form of enlargement of the lymphoid follicles which develop active germinal centres. These are characterised by numerous centroblasts and centrocytes, a rich admixture of macrophages with a poorly defined pale cytoplasm containing tingible bodies, and a surrounding cuff of small lymphocytes. Alternatively, there may be expansion of the interfollicular tissue by numerous mature lymphocytes, lymphoplasmacytoid cells, plasma cells and varying numbers of immunoblasts. A mixture of both patterns is present in some cases. Nodes draining tumours or other sources of tissue breakdown may be further expanded by the presence of numerous histiocytes in the sinusoids, a picture referred to as sinus histiocytosis.

Cytological findings: reactive hyperplasia (Figs 13.2, 13.3)

Non-specific hyperplasia

Non-specific hyperplasia yields a cytological pattern on FNA which depends on the proportions of follicular and interfollicular tissue in the aspirate, and this in turn usually correlates with the histological findings described above. Thus, smears from a node composed predominantly of large follicles with active germinal centres contain many centroblasts and centrocytes, while the interfollicular tissue is comparatively sparse and represented by mature lymphocytes, plasma cells and immunoblasts (Fig. 13.2).

Fig. 13.2 Reactive lymphadenitis. Mixed lymphoid cells, granulocytes, plasma cells and tingible body macrophages (MGG).

Fig. 13.3 Infectious mononucleosis. Several atypical immunoblasts are present in a background of small lymphocytes (MGG).

In extreme cases, the pattern may mimic a mixed lymphoma of centroblastic/centrocytic type. The presence or absence of tingible body macrophages is of little diagnostic value. Immunocytochemical evaluation of the lymphoid population may be the only way to resolve this diagnostic problem.

In contrast, when interfollicular tissue predominates, the smears are rich in lymphocytes, plasma cells, lymphoplasmacytoid cells and some immunoblasts. Such smears are difficult to differentiate from those of a low-grade lymphoma. Analysis of light chain immunoglobulin restriction is usually required to arrive at a conclusive diagnosis.

Some conditions lead to a cytological pattern which clearly deviates from the general types described above. A description of the best recognised of these follows. It is important to remember that definitive diagnosis is dependent on good clinical correlation.

Viral and post-vaccinial lymphadenitis

These conditions cause intense reactivity in the interfollicular tissue which presents with a prominent immunoblastic proliferation in addition to lymphoplasmacytoid cells and plasma cells.

HIV infection

Generalised lymphadenopathy is common in patients with acquired immunodeficiency syndrome (AIDS).³¹ There is a florid follicular hyperplasia with immature follicle centre cells in a background of mature lymphocytes, plasma cells and macrophages (Fig. 13.4). Immunoblasts are always present. The pattern is non-specific and thus not diagnostic for AIDS.

Fig. 13.4 HIV infection. Florid follicular hyperplasia with large number of immature follicle centre cells mixed with mature lymphocytes and one macrophage with tingible bodies (MGG).

The lymphoid cells are mostly polyclonal B cells but some mature T cells are also present.

Infectious mononucleosis

Infectious mononucleosis can also cause lymphadenitis, which is mainly confined to the interfollicular tissue (Fig. 13.3). Cytologically, it is characterised by numerous immunoblasts, some of which are atypical with large irregular nuclei.³² In rare cases the pattern may even be suggestive of Hodgkin’s disease.³² Serological tests can be helpful, but phenotyping of the atypical cell population may be the only way to rule out a lymphoma.

Rheumatoid arthritis, systemic lupus erythematosus and secondary syphilis

All of these conditions can cause massive lymphadenopathy. Cytologically, there is a reactive pattern with numerous plasma cells, some containing Russell bodies, which may lead to a suspicion of a low-grade lymphoma with plasmacytic differentiation. In such cases, it may be impossible to arrive at a conclusive diagnosis without resorting to immunocytochemistry.

Dermatopathic lymphadenopathy

This is a special variant of reactive lymphadenitis which is observed in patients with chronic skin disorders such as psoriasis or dermatitis. The germinal centres are hyperplastic and the interfollicular tissue is expanded by cells of histiocytic appearance.³³ Smears from such lymph nodes show numerous small lymphocytes, plasma cells, eosinophils and occasional blast cells. There are numerous histiocyte-like cells, also known as interdigitating reticulum cells, with pale indistinct cytoplasm. Macrophages containing brown melanin pigment from the damaged skin are always present.

Immunocytochemistry (Figs 13.5–13.7)

In reactive lymphadenitis the small lymphocytes are mostly T cells of which the helper type predominate. The B cells are of various sizes and are polyclonal, expressing both kappa and lambda light chains. Atypical immunoblasts are either of T or B phenotype, some of which may also express CD30 (Ki-1). They do not express CD15 (Leu M1). This phenotype is inconsistent with that of the neoplastic cells in Hodgkin lymphoma, where the mixed cellular infiltrate might be mistaken for a reactive picture.

Fig. 13.5 Immunocytochemistry in reactive lymphadenopathy. Cytospin material from the aspirate shown in Figure 13.2. B- and T-cell markers identify a mixed population of lymphoid cells (A). B cells of varying sizes (B). Small mature T cells (alkaline phosphatase).

Fig. 13.6 Immunocytochemistry in reactive lymphadenopathy showing polyclonal light chain expression with no predominance of either kappa or lambda to suggest lymphoma. (A) Kappa; (B) Lambda (alkaline phosphatase).

Fig. 13.7 8-colour flow cytometry analysis of fine needle aspirate from a lymph node with reactive follicular hyperplasia. The following antibody panel was used: Lambda FITC/ Kappa PE/CD19 PerCP-Cy5/ CD10 APC/ CD5 PE-Cy7/ CD4 APC-Cy7/ CD8 AM-Cyan/ CD3 Pacific Blue. Data were acquired using FACS-Canto and DIVA software (Becton Dickinson). Data analysis was done using Infinicyt (Cytognos, Salamanca, Spain) software. Upper row: Left: forward scatter/side scatter plot shows that most cells were in the lymphocyte area. Dead cells (yellow) are excluded from analysis. Middle: CD19 vs CD3 plot shows 47% B cells (red) and 43% T cells (blue). Right: Kappa (orange)/ Lambda (green) analysis in CD19/SSC gated B cells shows normal kappa/lambda ratio 1.3. Middle row: Left: CD19 vs CD5 plot within CD19/SSC gate shows a small population of CD5+ B cells that correspond to mantle zone B cells (5% of B cells). Middle: CD19 vs CD10 plot within CD19/SSC gate shows a somewhat larger population of CD10+ B cells that correspond to germinal center cells (20% of B cells). Right: CD3 vs CD5 plot shows that most T cells were positive for both markers (blue). Lower row: Left: Kappa/lambda analysis of CD5+ B cells shows normal kappa/lambda ratio (1.2). Middle: Kappa/lambda analysis of CD10+ B cells shows normal kappa/lambda ratio (1.5). Right: CD4 and CD8 expression in CD3-gated T-cell population. CD4/CD8 ratio was increased to 8.5.

(Courtesy Professor A. Porwit, Hematopathology Division, Dept Pathology and Cytology, Karolinska University Hospital Solna, Stockholm, Sweden)

The flow cytometry pattern of reactive lymphadenitis is seen in Figure. 13.7.

This is a very common finding in reactive lymph nodes and often associated with follicular hyperplasia but may also be seen in its absence. Characteristic is dilatation of subcapsular and trabecular sinuses, which are partially or completely filled with histiocytes/macrophages. This type of hyperplasia is observed in lymph nodes which drain areas with cancer as well as inflammatory lesions but in many cases the cause is unknown.

This is a rare, extreme form of sinus histiocytosis that was first described by Rosai and Dorfman in 1969.³⁴ The disorder is seen most often in black children and adolescents. Most patients are in good health and develop massive bilateral non-tender enlargement of the cervical lymph nodes followed by fever. Extra nodal involvement has also been described. The cause is unknown but the disorder has a prolonged course and spontaneous regression of the nodes usually takes place.

Cytological findings: sinus histiocytosis with massive lymphadenopathy (Fig. 13.8)

There are numerous lymphocytes and large pale histiocytes which have vesicular nuclei with small nucleoli and an abundant vacuolated cytoplasm. The histiocytes often have well-preserved lymphocytes in the cytoplasm which is referred to as lymphocytophagocytosis or emperipolesis.^35–38

Fig. 13.8 Rosai–Dorfman’s disease. Large histiocyte with prominent nucleoli and lymphophagocytosis (MGG).

Histiocytic necrotising lymphadenitis is a rare well-defined clinical entity which was first described by Kikuchi and Fujimoto et al. in 1972.^39,40 It affects mainly young women presenting with fever and enlargement of one or more cervical nodes. It is a benign, self-limiting disease and its aetiology is still unknown.

Cytological findings: Kikuchi’s disease (Fig. 13.9)

Numerous foamy macrophages as well as ‘tingible body’ macrophages containing karyorrhectic debris in a background of necrotic material. Small lymphocytes, as well as activated lymphocytes are found. Neutrophils, epithelioid cells and plasma cells, when present, are few in number.⁴¹

Fig. 13.9 Kikuchi’s lymphadenitis. Lymphocytes mixed with phagocytic histiocytes with vacuolated cytoplasm and irregular punched-out nuclei (MGG).

Lymph nodes draining or adjacent to a focus of bacterial infection, may be directly invaded by the organisms, causing acute lymphadenitis followed in some cases by suppuration. Initially a light infiltrate of neutrophil polymorphs is present but as the tissues undergo necrosis the node becomes a suppurative mass. Appropriate treatment may result in resolution or scarring.

This variant of infectious lymphadenitis occurs often in otherwise healthy children without clinical signs of infection. They present with a firm, often non-tender enlarged lymph node in the neck. The most common pathogen in non-tuberculous mycobacteriosis is Mycobacterium avium. ⁴²

Aspirated material is often purulent and it is important to recognise the need for mycobacterial culture.

Cytological findings: suppurative non-tuberculous mycobacteriosis (Fig. 13.10)

• Thick smear with cellular debris and granulocytes

• Variable amount of lymphocytes

• Few macrophages and epithelioid histiocytes

• Multinucleated giant cells are rarely seen.

Fig. 13.10 Suppurative non-tuberculous mycobacterial lymphadenitis. Cellular debris, neutrophil granulocytes, mature lymphocytes and epithelioid histiocytes in irregular clusters (MGG).

This systemic disorder of young adults is characterised histologically by the presence of non-caseating giant cell granulomata and tends to affect lungs and lymph nodes mainly but most organs can be involved. A similar reaction is sometimes seen in nodes draining a primary carcinoma whether or not metastases to the node have occurred. This finding is referred to as a sarcoidal reaction.

Cytological findings: sarcoidosis (Fig. 13.11)

• The aspirate contains cohesive clusters of epithelioid cells and numerous small mature lymphocytes

• In most cases multinucleated giant cells are present

• The background is free from necrosis, a finding strongly suggestive of sarcoidosis.

Fig. 13.11 Sarcoidosis. Cluster of epithelioid cells forming a granuloma (MGG).

Diagnostic pitfalls: sarcoidosis

If Langhans giant cells are absent the differential diagnosis should include Hodgkin’s disease and low-grade T-cell lymphoma. Techniques such as PCR and special stains for organisms such as mycobacteria and fungi, as well as immunocytochemistry to characterise the lymphoid cells are of value in reducing the number of diagnostic alternatives. In sarcoidosis, the lymphoid population is dominated by T cells, with a normal ratio of helper to suppressor cells, while the B cells are polyclonal.

Infection of lymph nodes by Mycobacterium tuberculosis is usually the result of spread from primary lung infection and can present clinically with massive generalised lymphadenopathy, especially of the cervical nodes, even to the extent of simulating lymphoma. The hallmark of tuberculosis histologically is the presence of caseating necrosis associated with epithelioid giant cell granulomata. Early diagnosis is particularly important since the condition is treatable.

Cytological findings: tuberculous lymphadenitis (Figs 13.12, 13.13)

Aspiration smears from tuberculous lymphadenitis show three major cell patterns:

• Epithelioid granulomas without necrosis, in which there are small clusters of epithelioid histiocytes and single forms, mixed with reactive lymphocytes, but Langhans giant cells are not often seen

• Epithelioid granulomas with necrosis, showing similar features, but in addition there is a variable amount of pale stained amorphous material in the background

• Necrosis without epithelioid granuloma. This type shows thin necrotic debris containing large numbers of polymorphonuclear cells and scattered histiocytes.

Fig. 13.12 Tuberculosis. A granuloma composed of epithelioid cells is present in a background of necrosis with numerous granulocytes (MGG).

Fig. 13.13 Tuberculosis. This field contains a mixture of epithelioid cells, plasma cells, lymphocytes and macrophages (MGG).

For definitive diagnosis, acid fast bacilli can be identified using the Ziehl–Neelsen (ZN) stain or other stains for acid fast bacilli.^43–45 These stains have a relatively low sensitivity and are nowadays mostly replaced by PCR techniques to identify the mycobacteria.

Immunodeficient patients, including those with AIDS, suffer from many of the infectious causes of lymphadenitis and are especially predisposed to tuberculosis, but may also be infected by less common organisms which are rarely encountered in the general population. Infection due to Mycobacterium avium intracellulare is an example of this type of lymphadenitis and is recognised with increasing frequency in this group of patients.

Histologically, the lymphoid tissue is replaced by large histiocytes with voluminous finely vacuolated, ill-defined cytoplasm containing numerous bacilli.

Cytological findings: atypical mycobacterial infection (Figs 13.14, 13.15)

• Smears show histiocytic cells with abundant pale cytoplasm

• In MGG-stained preparations the mycobacteria present as cylindrical non-stained ‘negative images’ of bacilli that are diagnostic of mycobacterial infection⁴⁶

• The presence of bacilli can sometimes be demonstrated using the ZN stain. Characteristically, they are arranged randomly within the cytoplasm.

Fig. 13.14 Atypical mycobacterial infection. Histiocytes with abundant ‘foamy’ cytoplasm are seen. In an immunocompromised patient this picture should arouse suspicion of an atypical mycobacterial organism (MGG).

Fig. 13.15 Ziehl–Neelsen staining of the aspirate shown in Figure 13.15, revealing numerous acid fast bacilli distending the histiocytic cells.

Leprosy is a chronic destructive systemic infection due to Mycobacterium leprae and is now mainly seen in third world countries. As in the histology of this disease, two different types of reaction are seen cytologically in affected lymph nodes, referred to as lepromatous and tuberculoid.⁴⁷

In the lepromatous or Virchow’s form of leprosy, the enlarged lymph nodes yield syncytial histiocytes with abundant clear cytoplasm containing numerous acid fast bacilli. In leprosy, the bacilli are present in parallel disposition in the form of globi in the cytoplasm of the histiocytes which are sometimes referred to as Virchow’s or globus cells. The arrangement of organisms is important in distinguishing leprosy from atypical mycobacterial infection.^47,48

In the tuberculoid form of leprosy, the predominant cytological picture is a granulomatous process, containing epithelioid histiocytes in a background of lymphocytes. Organisms are present in low numbers, and are more difficult to identify than in the lepromatous form.

Paracoccidioidomycosis (Figs 13.16, 13.17)

Paracoccidioidomycosis is a fungal infection endemic in South America. P. brasiliensis often causes massive lymphadenopathy which results from a granulomatous reaction. Epithelioid cells, multinucleated giant cells, neutrophils and eosinophils are found in varying numbers. The diagnosis is established by identification of multiple budding spores, 5–15 μm in diameter, with birefringent cell membranes.⁴⁹ A Gomori–Grocott silver stain will readily identify the spores.

Fig. 13.16 Paracoccidioidomycosis. Multinucleated giant cell and histiocytes in a necrotic background with granulocytes. The spores are seen as rounded structures with birefringent capsules (MGG).

(Courtesy of Dr RM Viero, Dept Pathology, Faculdade de Medicina de Botucatu UNESP, Brazil.)

Fig. 13.17 Paracoccidioidomycosis. (A) Multinucleated giant cell with spores visible in the cytoplasm (MGG). (B) Spores with pathognomonic multiple budding (Gomori).

(Courtesy of Dr RM Viero, Dept Pathology, Faculdade de Medicina de Botucatu UNESP, Brazil.)

Histoplasmosis (Fig. 13.18)

Histoplasma capsulatum is another fungal infection that can give rise to a granulomatous reaction. The yeast form is oval and 2–3 μm in diameter and resides in the cytoplasm of macrophages.

Fig. 13.18 Histoplasmosis. (A) Several histiocytes with tiny oval spores characteristically within the cell cytoplasm (Shorr). (B) Multiple spores identified by Gomori.

(Courtesy of Dr RM Viero, Dept Pathology, Faculdade de Medicina de Botucatu UNESP, Brazil.)

Cryptococcus neoformans is also one of the fungal infections that may lead to a granulomatous reaction in lymph nodes. However, most cases present with an inflammatory infiltrate dominated by neutrophils and histiocytes.

Actinomycosis, the condition caused by filamentous bacterial organisms of the Actinomyces species, is a further source of granulomatous inflammation to be considered in the differential diagnosis.⁵⁰ The organisms are best shown by Gram-stain.

Talc, silicone or beryllium can induce massive lymphadenopathy which is impossible to differentiate clinically from metastatic lymph node disease. The aspirated material consists mainly of giant cells containing foreign body particles, together with lymphocytes of mature type and mononuclear histiocytes.^51,52 Antibodies to vimentin, and epithelial, lymphoid, melanocytic and myogenic differentiation markers should be used to corroborate the diagnosis.

Malignant lymphomas are divided into two major categories: Hodgkin lymphoma and non-Hodgkin lymphomas. They can be further divided into several subgroups, which are important to identify because of their different clinical behaviour. Hodgkin lymphoma is most commonly subclassified according to the Rye scheme which was proposed in 1966,⁵³ which is also followed in the recent WHO classification.⁵⁴

The classification of non-Hodgkin lymphomas has been more controversial. The Kiel classification propounded in 1975 and the 1982 Working Formulation have been the two most commonly used schemes for this group of tumours.^55,56 Histological assessment of architectural and cytological features has traditionally formed the basis for all of the classifications. The updated Kiel classification, published in 1988, also incorporated data from immunophenotypic analysis.⁵⁷ In the REAL (Revised European-American classification of Lymphoid neoplasms) an attempt was made to define clinical relevant subgroups of lymphomas that could be recognised with available morphological, immunological and genetic techniques.⁵⁸ The ‘WHO classification of tumours of haematopoetic and lymphoid tissues’ is based on the same parameters as the REAL classification and is today generally accepted.⁵⁴

Much effort has been spent on the diagnosis of malignant lymphomas by FNA, attempts which have until recently been only partially successful. One major reason for this is that most neoplastic lymphoid cells lack the traditional cytological features of malignancy. Such cells are close replicas of their benign counterparts. In many instances the cytological diagnosis therefore rests on evaluation of whether or not the smears show a spectrum of cells in proportions typical of benign conditions.

If the FNA sample is composed of only one cell type a confident diagnosis of non-Hodgkin lymphoma can usually be made. However, some lymphomas are composed of several types of neoplastic cells, while others contain a confusing admixture of benign lymphoid cells with neoplastic elements, which obviously obscures the picture. The complexity of such samples may be an overwhelming task even for the most experienced cytopathologist. In the case of Hodgkin lymphoma the finding of cells with large atypical nuclei and multilobated nuclei has been considered diagnostic.

At present no system of classification has been constructed for FNA cytology material. From published data, it seems clear that a histological diagnosis based on the Kiel classification correlates very well with FNA findings.⁵⁹ This partly results from the fact that the Kiel classification has only two subgroups in which growth pattern is of importance for diagnosis and choice of therapy. In both the REAL and WHO classification systems there is a much greater emphasis on cytomorphology, immunophenotyping and molecular studies than on architecture growth pattern. Growth pattern, whether nodular or diffuse, contributes to diagnosis in only one subtype. Thus this system will allow a conclusive diagnosis and subtyping of most lymphomas on cytological material if the morphological evaluation is combined with immunophenotypic studies and sometimes cytogenetics.^60–62

Diagnosis and subclassification of Hodgkin lymphoma have been attempted on FNA material.^63–65 Again, the classification schemes in current use are based on both architectural and cytological features in excised tissue, making their application to FNA material somewhat difficult.

The reported accuracy of cytological diagnosis and classification of lymphomas on FNA samples varies between 10% –90%.⁶⁶ Not surprisingly, this degree of variation has impeded the acceptance of FNA cytology as the sole diagnostic modality in patients with suspected lymphoma. However, as previously pointed out, FNA cytology is more readily accepted for evaluation of patients with suspected recurrent lymphoma, or deep-seated primary lymphomas. This attitude is somewhat puzzling since the diagnostic difficulties encountered in these special circumstances are identical irrespective of the fact that the lymphoma is primary or recurrent, superficial or deep-seated.

The use of ancillary techniques including immunocytochemistry, cytogenetics and DNA hybridisation has greatly increased the utility of cytological material for conclusive diagnosis of lymphoma. In fact, cytology specimens seem ideal for immunological evaluation; recent studies show a high diagnostic accuracy if the cytological findings are combined with results from immunophenotyping and clonal restriction analysis.