Nomenclature used in reporting

Reporting of thyroid FNA specimens should follow a standard format that is clinically relevant in order to direct management. At the National Cancer Institute sponsored thyroid state of the science conference in Bethesda in October, 2007, consensus was reached regarding indications, pre-FNA requirements, FNA techniques, diagnostic terminology, etc.²⁵ The Bethesda System reporting terminology includes six categories: non-diagnostic, benign, atypia of undetermined origin, FN/suspicious of FN, suspicious for malignancy and malignant.²⁶ Every category carries with it the implied risk for malignancy. Each category should be further qualified as to the possible pathological entity.

If an indeterminate diagnosis is being made due to features suspicious but not diagnostic of a neoplasm, it should be so qualified, since repeat FNA may enable definitive diagnosis. If, on the other hand, it is being made for FN, qualifying it as such will clarify that distinction of benign from malignant cannot be achieved by repeat FNA, and either ancillary techniques or histological study are required. To simplify the issue, we suggest that in the former, a diagnosis of ‘indeterminate (suspicious)’ be given and in the latter ‘indeterminate (FN)’. We suggest the revised Papanicolaou system of reporting¹⁰ which is simple and easily reproducible with the following six categories that are useful in triaging patients for either clinical follow-up or surgery:

Accuracy and limitations of cytodiagnosis

In experienced hands, and in situations where the pathologist performs the needling, cytology can be a very sensitive tool with sensitivity and specificity of up to 94% and 98% for the diagnosis of malignant lesions¹⁰ and nearly 90% accuracy rates for the identification of malignancy if follicular lesions are excluded.^27,28 Cytologic diagnosis is generally accurate in thyroiditis, usual type of PC, medullary carcinoma (MC), anaplastic carcinoma (AC) and high-grade lymphoma. False negatives generally occur in cystic lesions harboring malignancy, in low-grade or intermediate-grade lymphomas occurring in a background of Hashimoto’s thyroiditis (HT), in AC with necrosis, in focal involvement of the gland by thyroiditis and in cases with dual pathology where the dominant non-neoplastic lesion overlies or obscures a small carcinoma.^29–31 False negatives have been shown to be minimized by using US-guided FNA. The false-positive rate can be reduced further by excluding indeterminate follicular lesions.

A satisfactory sample

While abundant colloid without altered blood or debris usually indicates a benign lesion, the presence of intact and well-fixed follicular cells is obligatory for a smear to be considered as satisfactory. Relaxing the criteria for a satisfactory sample often leads to higher rates of false-negative diagnoses. Many false-negative diagnoses in thyroid cytology are related to poor-quality specimens being reported as nonmalignant.⁶⁵ A cytological sample from a benign thyroid nodule can be considered satisfactory if six clusters of benign cells are seen in at least two slides prepared from two needle passes.⁶⁶ This criterion can be softened in situations where the pathologist performs the procedure. Two to three needle passes, performed gently and with small-caliber needles, are usually accepted easily by the patient. Increased numbers of needle passes improve the diagnostic rate but often at the cost of patient discomfort. US-guided procedures can greatly diminish non-diagnostic rates, especially when radiologist and pathologist work together.

Normal structures

Follicular epithelial cells and colloid are regular features in normal thyroids and in colloid goiter. Follicular cells show fragile gray-blue or pale-blue cytoplasm with indistinct or fuzzy cell borders. Coarse blue (paravacuolar) cytoplasmic granules may be seen (Fig. 6.1). Bare nuclei, similar in shape and size to normal lymphocytes, are common. Some cells may show small nucleoli.

Fig. 6.1 Follicular epithelium

Uniform cells with fragile, partially disrupted cytoplasm; bare lymphocyte-like nuclei in a background of thin colloid (MGG, HP).

In non-bloody specimens, thin colloid stains blue, violet or pink and forms a thin membrane-like coating or film, with folds and cracks due to drying of colloid on the slide (Fig. 6.2A,B). Colloid may wash off from the slide while staining but the parched-earth or crazy-pavement artifact of colloid remains and follicular cells are often seen at the smear margins. Thick colloid appears as round, dense clumps of deep blue, violet or magenta-colored acellular material, or as globular masses with superimposed follicular cells, especially in samples from NG. Colloid can be mistaken for hyalinized collagenous stroma (collagenous spherules) or amyloid. Skeletal muscle fragments appear as straps of dark-blue material with pale ovoid nuclei and cross-striations visible in higher magnification. In PAP-stained smears, thin colloid stains pale green or orange, with cracking artifacts seen. Thick colloid appears as clumps of dark green or orange material (Fig. 6.2C,D). The blue violet color and hyaline texture of colloid appear to advantage in MGG-stained smears and distinction from fibrillary collagen and deep magenta staining amyloid is easier (Fig. 6.3). In bloody smears, colloid resembles other protein-rich fluids, including serum.

Fig. 6.2 Colloid

(A, B) Thin colloid forms a varnish-like coat of relatively homogenous material, characteristic ‘crazy pavement’ and cracking artifacts (A, MGG IP; B, Pap, IP); (C, D) Thick colloid forms irregular dense clumps of material; homogeneous and violet in MGG; variable density and staining in Pap. Compare with Figure 6.3 A and B (C, MGG, IP; D, Pap, IP).

Fig. 6.3 Collagen and amyloid

(A) Fragment of collagenous stroma; fibrillar texture; red staining; a few nuclei included (MGG, HP); (B) Clump of amyloid in medullary carcinoma; structureless but not homogeneously hyaline; magenta color (MGG, HP oil).

C-cells resemble medullary thyroid carcinoma cells and need immunocytochemical stains for identification, except in C-cell hyperplasia, where they are present in large numbers. Accidental puncturing of the trachea or larynx during FNA can be suspected if the patient coughs and air enters the syringe. Smears show mucus, respiratory cells and carbon-laden macrophages. Cartilage may be seen, appearing as brilliant magenta flecks with fibrillary edges.

Simple colloid goiter

A diffusely enlarged gland with smears showing a normal cytological appearance (or abundant or very thick colloid) indicate a simple colloid goiter which is an early stage in the evolution of NG.

Nodular goiter (Figs 6.4–6.7)

Fig. 6.4 nodular goiter

Follicular epithelial cells, some of hyperplastic type with abundant fragile cytoplasm, some of involutional type with small, dark, mainly naked nuclei, background of thin colloid (A, MGG, HP; B, Pap, HP).

Fig. 6.5 nodular goiter

Cystic degeneration: involutional follicular epithelium, foamy macrophages, background of blood and thin colloid (A, MGG, HP; B, Pap, HP).

Fig. 6.6 nodular goiter

(A) Flat monolayered sheet of epithelial cells representing a flattened macrofollicle; sheet has frayed edges, honeycomb structure (Pap IP); (B) Intermediate-size follicle seen as a three-dimensional ball of cells confined by a basement membrane (MGG, HP).

Smears show abundant thick or thin colloid, follicular cells in monolayered sheets, poorly cohesive groups and as single cells, globular colloid masses with superimposed follicular cells, bare nuclei and pigment-laden histiocytes (foam cells) in varying proportions. Involutional follicular cells with small round dark nuclei and fragile, feathery cytoplasm as well as larger, hyperplastic cells with abundant vacuolated cytoplasm or with marginal vacuoles (fire-flares) are seen (see Fig 6.9). The latter may show anisonucleosis. Oxyphilic (Hurthle) cells may be seen.

Macrofollicles disrupted by needling flatten on the slide to form monolayered sheets of epithelial cells. These have a honeycomb structure due to distinct cell membranes (Fig. 6.6A)³ and frayed edges. Focally, the cytoplasm is indistinct, forming a web-like background to the nuclei. Smaller follicles may be removed intact by the needle. They appear as spherical cell clusters resembling multinucleate giant cells (Fig. 6.6B), that may be enveloped by a basement membrane. Macrofollicles are evidence of benignity and are of diagnostic significance. Hyperplastic papillae containing follicles and intact dilated follicles in cell-block preparations are supportive evidence of a benign nodule. Foam cells, often hemosiderin-laden, suggest degeneration, commonly seen in NG (Fig. 6.5A). Distinction between degenerate epithelial cells and true macrophages is not always possible as transitional forms occur that show epithelioid as well as histiocytoid features and focal atypia. Hyalinized stroma presents as irregular pink/red frayed fragments of vaguely fibrillar material, some with adherent epithelial cells (see Fig. 6.3A).

Inadequate samples may be obtained from colloid nodules due to low cellularity and degenerative change. If smears contain few or no well-preserved follicular cells, the specimen should be reported as non-diagnostic, unsatisfactory, and repeated.

The cytological appearances of NG can overlap with FN and cytological criteria alone cannot always reliably distinguish between the two. Selective sampling of a microfollicular focus in NG leads to a repetitive pattern of microfollicles or rosettes with no colloid, and distinction from FN may be impossible.⁶⁷ However, since this is a focal phenomenon, samples from other areas are likely to show macrofollicles, abundant colloid and degenerative changes recognizable as colloid goiter. In the cytological spectrum of follicular nodules, a large sheet pattern of follicular cells with thin colloid in the background indicates a macrofollicular pattern suggestive of benignity while syncytial clusters with nuclear crowding and overlapping suggest a neoplasm.⁶⁸

Cystic PCs often contain abundant colloid. This can cause diagnostic difficulties if smears are poor in cells, but a close look at the nuclear features should allow a correct diagnosis in most cases, as detailed below. Smears in FV-PC may show well-formed follicles containing colloid.

Groups of large cells with irregular nuclei, not infrequently found in NG, are probably related to degenerative change. Their origin is uncertain; they may be histiocytes or regenerating epithelial cells consistent with repair (Fig. 6.7). Prominent aggregates of histiocytes can in some cases mimic cells of PC due to similar nuclear features.⁶⁹

Fig. 6.7 Histiocytes in nodular goiter

Histiocytes with abundant cytoplasm and enlarged, irregular nuclei with distinct nucleoli; these could be mistaken for atypical epithelial cells; bare nuclei of degenerate follicular cells in the background (MGG, HP).

The value of cytology in hyperfunctioning nodules has been disputed but recent reports suggest that there may be an increased incidence of malignancy in hyperthyroidism and that FNA is a reliable diagnostic method in these cases. It appears reasonable, therefore, to evaluate these lesions cytologically prior to radioactive iodine treatment or surgical intervention.⁷⁰

Cystic nodules

Thyroid cysts are most commonly due to retrogressive changes in NG where they may be small, yielding a few drops of fluid or larger cysts yielding substantial quantities. FNA yields brownish colloid-like fluid with altered blood. Smears show foam cells that may contain hemosiderin and sparse degenerating follicular epithelium (see Fig 6.5).^31,71

Cystic change and/or hemorrhage occur in thyroid tumors (25% of PCs, and 20% of FNs in one series).⁷² Prevalence of malignancy in resected cystic nodules is 10–15%.^4,31 Partly solid and cystic nodules may also harbor malignancy.

A definitive diagnosis of cystic NG requires adequate sampling of any solid component. Cystic fluid containing only macrophages and no epithelial cells does not rule out a cystic neoplasm. The gross appearance of cyst fluid is not helpful.³¹ Presence of atypical cells in a ‘cyst fluid only’ sample should lead to a ‘suspicious’ diagnosis (not non-diagnostic).⁷³ A cystic lesion that can be completely evacuated with no palpable nodule remaining and no epithelial atypia indicates benignity.⁷¹ While 4–40% of cystic lesions can be permanently cured by FNA, others need repeated evacuations. False-negative cytological diagnosis is most common in cystic carcinomas, especially PC, where only up to 60% can be correctly diagnosed.^71,74 Re-biopsy of the cyst bed and of any residual or recurrent swelling, preferably with US-guidance, is advisable. Recurrent cysts, lesions greater than 3–4 cm in diameter and lesions in young males are indications for surgical excision.

Thyroglossal cysts yield clear or mucoid fluid and smears may contain squamous or respiratory epithelium associated with colloid.⁷⁵ Foregut cyst of thyroid yields yellowish fluid and smears show detached ciliary tufts and macrophages.⁷⁶ Rare epidermoid cysts of thyroid have been described.⁷⁷

Clear fluid aspirated from a lateral cystic lesion suggests a parathyroid cyst and parathormone estimation of the fluid is advised.⁷⁸ Thymic cysts yield clear fluid containing lymphocytes.

Acute suppurative thyroiditis

This is an uncommon but potentially life-threatening condition⁷⁹ occurring mostly in debilitated or immunosuppressed individuals.⁴² Patients present with extremely tender thyroid enlargement, fever and high ESR. Smears show neutrophils, necrotic cells and debris. Intracellular bacteria, (usually Gram-positive cocci), may be present. Less commonly, mycobacteria, viruses, aspergillus, actinomycosis, cryptococcosis and pneumocystis have been observed.⁴² Cytologic material can also be sent for culture and sensitivity.

Autoimmune thyroid disease

The syndromes comprising autoimmune thyroid disease are many intimately related illnesses, the two most common being Graves’ disease (GD) (with goiter, hyperthyroidism and, in many patients, associated ophthalmopathy) and Hashimoto’s thyroiditis (HT) (with goiter and euthyroidism or hypothyroidism). Immunological mechanisms in these diseases are closely related and the syndromes are connected together by similar thyroid pathology, co-occurrence in family groups, and transition from one clinical picture to another within the same individual over time. Antibodies to thyroid peroxidase (TPO-Ab), produced mainly by intrathyroidal lymphocytes, are the hallmark of autoimmune thyroid disease and are present in most patients with HT and in 75% of patients with Graves’ hyperthyroidism.⁸⁰

Graves’ disease (primary hyperplasia) (Figs 6.8 and 6.9)^81,82

Diagnostic criteria

♦ Colloid-free bloody background,

♦ Moderate to high cellularity,

♦ Follicular cells in monolayered sheets, follicular or ring structures,

♦ Moderate amounts of pale, cobweb-like, delicately vacuolated cytoplasm,

♦ Marginal vacuoles (fire flares).

Cytological study is not usually sought in GD as the clinical and biochemical profile are characteristic in most cases. However, cytology aids distinction from other conditions that present with thyrotoxicity, such as toxic NG, de Quervain’s thyroiditis, HT presenting in toxic phase and rarely in thyroid carcinomas.⁷⁰

Smears are bloody with scant colloid. Cellularity is moderate to high with follicular epithelium present as monolayered sheets, rings or follicular structures with suggestion of columnar shape (Fig. 6.8A). Cytoplasm is abundant and cobweb-like and delicately vacuolated, with larger marginal vacuoles giving a characteristic ‘fire-flare’ appearance.⁸² ‘Fire-flares’ are pale pink/red clumps of material measuring 1–7 µm in diameter, often with a pale center, mainly seen at the rim of the cytoplasm around the edges of aggregates of follicular cells (Fig. 6.9). They may correspond to colloid droplets seen ultrastructurally or to dilated cisternae of endoplasmic reticulum, and are indicative of cellular hyperactivity. They are present in toxic NG, and may be seen in a smaller percentage of cells in HT, diffuse or nodular goiter, and occasionally in neoplasms, including carcinoma.^83,84 In untreated GD, however, up to 100% of cells may show fire-flares. Paravacuolar granules may be seen. There may be moderate to marked nuclear atypia, especially in cases treated with radioactive iodine or neomercazole (Fig 6.8B).⁸⁵ Papillary structures may be seen, occasionally resembling PC. Hurthle cells and lymphocytes and rarely multinucleate giant cells or epithelioid cells may be seen.⁸²

Fig. 6.8 Hyperplasia

(A) Clusters of hyperplastic epithelial cells with a follicular arrangement; abundant pale vacuolated cytoplasm and suggestion of columnar shape; (B) Striking anisokaryosis/nuclear atypia in a case of medically treated GD of long duration (MGG, HP).

Fig. 6.9 Marginal vacuoles/’fire flares’

Loose sheet of hyperplastic follicular cells with abundant cytoplasm and relatively large nuclei; note clumps of homogeneous pale pink material, so-called ‘fire flares’ around the periphery of the sheet; no clinical evidence of hyperthyreosis in this case (MGG, HP).

Problems and differential diagnoses

Dilution of cells by blood may result in paucicellular, non-diagnostic smears. The bloody, colloid-free, highly cellular cytological appearance in GD with nuclear atypia may be mistaken for a neoplastic lesion, especially in the absence of proper clinical details. The cytoplasm in cells of GD is very delicate and shows a cobwebby, lace-like appearance, unlike the denser cytoplasm and better-defined cell margins seen in neoplastic lesions, especially PC.

Autoimmune thyroiditis (Hashimoto’s thyroiditis/lymphocytic thyroiditis) (Figs 6.10–6.13)^81,86–89

Fig. 6.10 Hashimoto’s thyroiditis

Aggregates of oxyphil cells; background of blood and lymphocytes; note abundant cytoplasm, anisokaryosis and prominent nucleoli in A, more numerous lymphoid cells in B (A, MGG, HP; B, Pap, HP).

Fig. 6.11 Hashimoto’s thyroiditis

(A) Syncytial cluster of oxyphil cells; abundant cytoplasm; prominent anisokaryosis; (Pap, HP oil); (B) Poorly cohesive hyperplastic cells showing oxyphil transformation; some resemblance to histiocytes; adherent lymphocytes (Pap, HP).

Fig. 6.12 Florid lymphocytic pattern of HT, 2 cases

(A) Polymorphous population of lymphoid cells with high lymphoid : epithelial cell ratio (follicular cells in top center, MGG, IP); (B) Cellular smears, mainly reactive lymphoid cells, cluster of follicular cells in centre (MGG, HP).

Fig. 6.13 Hashimoto’s thyroiditis

Plump epithelioid-like oxyphil or histiocytic cells forming a granuloma-like cluster (MGG, HP).

Diagnostic criteria

♦ Lymphoid and plasma cells in the background,

♦ Lymphoid cells impinging on follicular cells,

♦ Lympho-histiocytic collections,

♦ Hurthle cell change (variable),

♦ Multinucleate giant cells and epithelioid cells (variable).

Lymphocytic thyroiditis and HT represent different phases or manifestations of an organ-specific immunologically mediated inflammatory disease. A defect in suppressor T cells makes the gland vulnerable to cytotoxic T cells and stimulates T-helper cells to induce autoantibody production.⁹⁰ Patients usually present with diffuse or nodular thyroid enlargement and altered thyroid function caused by gradual immunologically mediated destruction of the gland. Antithyroid antibodies (especially microsomal/TPO-Ab) are significantly elevated in most cases. HT is one of the major manifestations of autoimmune thyroid disease, the other being GD.⁹¹ It is more common in Asians.⁹² In multiethnic Malaysia, a study of 88 cases of HT showed it to be more common in Indians than Chinese or Malays, and nodular presentation was seen in about one-third of cases.⁹³

A bloody background with lymphoid cells, degenerative changes in follicular cells and infiltration of follicular cells by lymphoid cells are characteristic features of HT. Variable features include oxyphilic cells (Hurthle cells), plasma cells, epithelioid cell granulomas and multinucleated giant cells.

The ‘lymphocytic’ pattern of HT occurs in children and young adults with a shorter history of the disease and absent or low antibody titers.⁸⁸ Smears are dominated by a mixed population of lymphoid cells including centroblasts, immunoblasts and dendritic reticulum cells from germinal centers characteristic of a reactive lymphoid proliferation (Fig. 6.12). Germinal center histiocytes have plentiful pale cytoplasm and oval or indented histiocytoid nuclei with granular chromatin. They are often clustered and associated with lymphoid cells, some of which lie within their cytoplasm. Histiocyte-lymphocyte rosettes or lympho-histiocytic clusters may be seen. Lymphoid : follicular cell ratios are often as high as 10 : 1 with epithelial cells so inconspicuous that smears resemble reactive lymphoid hyperplasia (Fig 6.12 A).

‘Classic’ or ‘florid’ HT occurs in older patients (usually women), who are more often hypothyroid and have raised TPO-Ab. The smear background shows lymphocytes with a variable number of plasma cells. There is prominent oxyphilic (Hurthle cell/Askanazy cell) change with single and syncytial aggregates of cells showing abundant, dense, finely granular, gray-blue cytoplasm (MGG), and well-defined cell borders. Nuclei are 2–4 times the size of normal follicular cell nuclei (Figs 6.10 and 6.11) and may show atypia and prominent nucleoli. Normal-appearing follicular cells may be present, showing features of hyperactivity. A characteristic feature is that of lymphocytes (and occasionally plasma cells) seeming to adhere to or infiltrate follicular cells, supporting the theory of direct epithelial damage by lymphocytes. Multinucleated giant cells and epithelioid cells can be seen in up to 40% of cases.^87,93 Neutrophils and eosinophils may be seen adhering to or infiltrating follicular cells in early stages.^42,93

Seven to thirty-three percent of cases are antibody negative.^87,90,93 In a study of 150 cases of HT, overall Ab positivity was found to be 88.67%.⁹⁴ TPO-Ab showed significant correlation with high lymphoid : epithelial ratios but not with cases showing follicular hyperplasia or hashitoxicosis (see below). It appears that antibody positivity may depend on the phase of the disease. Absence of serum antibodies can also be explained on the basis of local antibody production by intrathyroidal lymphocytes.⁹³

Problems and differential diagnoses

♦ Distinguishing bare thyroid nuclei from lymphocytes,

♦ Lymphoid populations in other lesions,

♦ Hyperthyroiditis/hashitoxicosis,

♦ Distinction from lymphoma,

♦ Distinction from follicular and Hurthle cell neoplasms,

♦ Distinction of Hurthle cells from other large nonlymphoid cells,

♦ Granulomatous thyroiditis (GT),

♦ Distinction from fibrosing variant of HT,

♦ Psammoma bodies.

Stripped follicular cell nuclei resemble lymphocyte nuclei in size and shape. However, they have more homogenous chromatin and denser nuclear rim and lack the basophilic rim of cytoplasm seen in lymphocytes. Smears in HT show a polymorphous population of lymphoid cells (including mature and transformed lymphocytes) with lymphoglandular bodies in the backgound.

Lymphoid cells are often seen in smears from PC, especially the diffuse sclerosing and Warthin-like variants.^95,96 Multiple sampling is important to ensure representative sampling so that neoplastic cells are not missed. Histological sections from thyroids resected for NG often show focal collections of lymphoid cells or lymphoid follicles. This does not imply a diagnosis of thyroiditis and review of cytologic smears from such cases rarely show lymphoid cells.⁸¹

Some cases of HT may present in a hyperthyroid state with increased T3 and T4 levels (hashitoxicosis).⁸⁷ In these cases, TPO-Ab are often negative.⁹⁴ Smears are highly cellular with hyperplastic follicular cells showing fire-flares. Lymphoid and Hurthle cells may be few or focal, and distinction from GD is often difficult or impossible. Six monthly follow-up of these cases with cytological monitoring and thyroid function tests have shown the patients becoming euthyroid within a few months to 2 years, with concurrently changing smear pattern to reflect the usual features of HT.⁴² Awareness of the close kinship between various forms of autoimmune thyroid disease (GD, HT and postpartum thyroiditis – described later) facilitates diagnosis and management.

While high-grade non-Hodgkin’s lymphoma is easily identified on cytological preparations, low-grade and MALT lymphomas are often difficult to distinguish from reactive lymphoid populations seen in thyroiditis. Approximately 75% of lymphomas arise in a background of HT. Focal involvement can cause sampling problems. The smear pattern may be of HT in one part of the gland, that of obvious lymphoma in another (see Fig. 6.65). The ‘florid lymphocytic’ type of thyroiditis with scant epithelial cells, common in young patients, should be viewed with suspicion if found in elderly individuals, and efforts made to rule out lymphoma. Flow cytometry and molecular assessment of lymphoid infiltrates in fine needle samples are being increasingly used for the distinction of lymphoma from thyroiditis.⁹⁷

Follicular and Hurthle cells in HT may show atypia significant enough to cause concern to the inexperienced observer (Fig. 6.11A). Sometimes, and more often in younger patients with florid lymphocytic thyroiditis, abundant active-looking epithelium may be aspirated, leading to a suspicion of neoplasia. While Hurthle cell atypia in a background of lymphoid cells is recognized as part of the diagnostic spectrum of the disease, selective sampling of lesions of focal nodular hyperplasia constituted by atypical Hurthle or follicular cells (seen in early phase of some cases of HT) with scanty or no lymphoid populations can be easily mistaken for neoplasia.^42,87 In so-called ‘burnt-out’ HT only oxyphilic cells may be present in smears.^89,98,99 This problem is more pronounced when such a case has a nodular presentation. Multiple sampling offers the best chance of finding evidence of lymphoid infiltration. Disorganized, poorly cohesive masses of oxyphilic cells with prominent nucleoli are more indicative of neoplasia, sheet-like structures infiltrated by lymphocytes of hyperplasia.¹⁰⁰ However, this distinction is not always obvious. Some authors advise against making a diagnosis of follicular/oxyphilic neoplasm in the presence of pleomorphism and abundant lymphoid cells.^82,101,102 The possibility of a neoplasm with surrounding HT should be considered if abundant epithelium and lymphocytes are aspirated.^98,101,103 Coexistence of HT with differentiated thyroid carcinoma (4%) or lymphoma (1%)⁴² may have to be considered, especially in cases with nodular presentation, observed in 22–80% cases in various series.^104,105

Many ‘hypertrophic epithelial cells’ with abundant cytoplasm are present in early stages of thyroiditis. These lack the dense cytoplasm and well-defined cytoplasmic borders of Hurthle cells. Some may even have ‘fire flares’ and presumably represent TSH-stimulated cells. Epithelioid-like cells with elongated, spindle-shaped cytoplasm and elongated or bean-shaped nuclei may be seen. These may resemble oxyphilic cells and forms morphologically intermediate between these two types occur (Fig. 6.13). Histiocytes or dendritic reticulum cells from germinal centers could be confused with oxyphilic cells, but lack the characteristic cytoplasmic density.

Cases of HT showing giant cells and/or epithleioid cells⁹³ may be confused with granulomaotus thyroiditis (GT).⁸¹ The inflammatory infiltrate in GT is not uniformly lymphocytic but is mixed, with evidence of more severe tissue destruction. The smear pattern is dominated by multinucleated giant cells surrounding extruded colloid and epithelioid cell collections. A few cases have showed overlap in cytological features with HT,¹⁰⁶ and thryoid function tests and antibodies are required to clarify the diagnosis.

Fibrosing variant of HT may be confused with Riedel’s struma,⁴² a rare thyroid manifestation of systemic collagenitis occurring in euthyroid middle-aged women. Smears yield paucicellular material containing collagenous fragments, bland or plump spindle cells and myofibroblasts.¹⁰⁷

Psammoma bodies have been described in association with HT¹⁰⁸ and in other benign processes. Their presence in smears probably warrants surgical biopsy because of close association with PC. Association of HT per se with PC is also reported.¹⁰⁹

de Quervain’s thyroiditis (subacute thyroiditis; granulomatous thyroiditis) (Figs 6.14–6.17)^42,81,87

This is a spontaneously remitting granulomatous inflammation of the thyroid, of possible viral etiology, occurring predominantly in females from the second to the fifth decades. Patients usually present with chills, fever, fatigue and a painful tender goiter that may be unilateral or spread from one lobe to the other.

Smears in GT are dominated by the presence of large multinucleate giant cells with numerous nuclei, granulomatous aggregates of epithelioid cells, degenerating follicular cells, neutrophils, macrophages and lymphocytes in a dirty smear background that shows debris and colloid.

The follicular cells often show degenerative features and contain dark-blue (golden in PAP-stained smears) cytoplasmic ‘paravacuolar’ granules representing lipofuscin or lysosomal debris. These granules are not a specific feature of this condition and may be seen in involutional follicular cells of NG, in GD and occasionally in PC and FNs.¹¹⁰ Stripped or crushed nuclei may be present. The giant cells are the hallmark of the disease and are characteristically very large, containing up to 200 nuclei (Figs 6.14 and 6.15), but even without them the diagnosis may be suggested if the other features of the disease are present (Fig. 6.16). Hurthle cells may be seen in some cases.

Fig. 6.14 de Quervain’s thyroiditis

Large multinucleated giant cells; clumps of thick, engulfed colloid (MGG, IP).

Fig. 6.15 de Quervain’s thyroiditis

Huge multinucleate histiocytic giant cells, dirty background of clumps of colloid, inflammatory cells and degenerate epithelial cells (Pap, IP).

Fig. 6.16 de Quervain’s thyroiditis

Mixed cell reaction; histiocytes, degenerating epithelial cells and lymphocytes in a ‘dirty’ background with thin colloid (MGG, HP).

The overlap with cases of HT showing multinucleate giant cells and granulomas has already been discussed. Epithelioid and giant cells may rarely be seen in GD. Giant cells are often seen in PC in association with lymphocytes and therefore PC must be considered in the differential diagnosis.¹¹¹ Rarely, the thyroid may be the site of mycobacterial infection, sarcoidosis or other infectious granulomatous processes.⁴²

Cases showing Hurthle cells may be confused with HT.¹⁰⁶

A small follicle withdrawn intact may simulate a multinucleate giant cell. The spherical nature and distinct outline of the structure in contrast with the irregular, flat form of true histiocytic giant cells should prevent confusion (Fig. 6.17A).

Fig. 6.17 de Quervain’s thyroiditis

(A) ‘Pseudogiant cell’; a small follicle in a smear from NG with a smooth well-defined border and a small amount of colloid in the center; superficial resemblance to a multinucleated giant cell (MGG, HP oil); (B) Degenerating epithelial cells with paravacuolar granules (left) and epithelioid histiocytes (right) in de Quervain’s thyroiditis (MGG, HP).

Cases with nodular presentation may simulate a lymph node or a neoplasm. Conversely, carcinomatous involvement of thyroid can mimic GT^112,113 or simulate silent thyrotoxic thyroiditis (hyperthyroiditis).

Characteristic cytological features of GT may be absent in the resolving phase of the disease.

Vigorous palpation of the gland may cause extrusion of follicular colloid, inciting a giant cell reaction. These granulomas are scanty, minute and clinical and functional profiles of GT are absent.¹¹⁴

Silent thyrotoxic thyroiditis (painless thyroiditis, subacute lymphocytic thyroiditis)^88,115,116 usually occurs in women as a sporadic or post-partum condition (post-partum thyroiditis), presenting with a small, diffuse, painless goiter. The disease may go through hyperthyroid, euthyroid, hypothyroid and recovery phases similar to GT. Low titers of TPO Ab are transiently present in two-thirds of cases. Smears show scattered lymphoid cells and giant cells in occasional cases. Unlike GT, there is no pain or any evidence of preceding viral infection and granulomas are uncommon. Rare cases have shown cytologic similarity to HT.¹¹⁶

Follicular neoplasms (Figs 6.18–6.24)^{23,29,68,117–120}

Fig. 6.18 Follicular neoplasm

Cellular smears of single cells, microfollicles or rosettes in a repetitive manner; benign adenoma by histology (A, MGG, HP; B, Pap, HP).

Fig. 6.19 Follicular neoplasm

Smears very similar to Figure 6.18; follicular carcinoma with vascular invasion by histology (A, MGG, HP; B, Pap, HP).

Fig. 6.20 Follicular neoplasm

Microfollicular cell clusters/rosettes; some nuclear hyperchromasia and coarseness in both (A) follicular adenoma and (B) follicular carcinoma (MGG, HP oil).

Fig. 6.21 Follicular neoplasm

(A) Small intact follicles with basement membrane; small uniform nuclei; follicular adenoma. (B) Microfollicular groups; enlarged nuclei; drop of abnormal colloid in small central lumen; follicular carcinoma (MGG, HP).

Fig. 6.22 Follicular neoplasms

Rationale for cytological diagnosis.

Fig. 6.23 Follicular carcinoma

Aspirate of distant lymph node metastasis of disseminated follicular carcinoma; syncytial cluster with some rosetting; bland nuclear chromatin; mild nuclear enlargement and anisokaryosis (MGG, HP).

Fig. 6.24 Atypical adenoma and well-differentiated follicular carcinoma

(A) Smear from atypical adenoma with bizarre cells; histology showed similar epithelial atypia but no capsular or vascular invasion; uneventful follow-up; (B) Well-differentiated follicular carcinoma metastatic to bone – cellular smear with prominent microfollicular pattern, relatively uniform nuclei and fire-flares (MGG, HP).

FNs are classified as benign (FA) and malignant (FC). FAs and most FCs are encapsulated tumors, occurring in one of the lobes. Histological diagnosis of a well-differentiated FC requires demonstration of capsular and/or vascular permeation. Most FNs, especially adenomas, have a uniform internal structure that is reflected in the cytological smears. FAs are more common in women and microscopically show a variety of histological patterns such as microfollicular (fetal), normofollicular, macrofollicular, trabecular, solid (embryonal), Hurthle cell and atypical adenomas.⁴² Cytologically, follicular lesions include FA, FC, cellular NG and FV-PC.¹²¹

Smears in FN are cellular in a bloody background that is usually devoid of colloid. Many uniform-sized follicular cell clusters, microfollicles and rosette formations are present. Syncytial aggregates, nuclear crowding and overlapping are also often seen.

The repetitive smear pattern with uniform cell population is in contrast to the variable pattern of different cell types seen in colloid and hyperplastic nodules. Microacinar clusters with a central lumen (that may contain a drop of colloid) represent microfollicles (Figs 6.18, 6.19 and 6.21B). These are characteristic of FN but may be found focally in NG. Rosette-like groupings without a lumen (Fig. 6.20) suggest a more solid growth pattern. A trabecular pattern is represented by rows and elongated aggregates of epithelial cells that resemble papillary structures when they adhere to strands of vascular stroma (see Fig. 6.33B,C). Small blood vessels with adherent epithelial cells can be found in any type of follicular neoplasm (see Fig. 6.25A).

The distinction between FN and NG is the most common differential diagnostic problem in solitary nodules as cytological appearances overlap (Fig. 6.22). A microfollicular focus in a colloid nodule cytologically resembles a microfollicular neoplasm, while smears from a macrofollicular (colloid) adenoma resemble a dominant nodule in multinodular goiter. Jaffar¹²² indicated that the presence of hemosiderin within macrophages and follicular cells excludes FN. The false-negative rate of cytology in FN may be 30% or more because of the inability to recognize normofollicular neoplasms.¹²³ However, these distinctions are of little clinical importance as long as the nodule is recognized as benign and spared from unnecessary surgery.

Most FCs are microfollicular, trabecular or solid, contain little colloid, and will be reported as ‘FN’ by cytology. Although the failure to recognize FC as neoplastic has been surprisingly high in some series,⁷ other studies show high diagnostic sensitivity, with false-negative rates as low as 0–2%.^11,13,28

Cytological features in FA and FC are similar, with cellular smears composed of syncytial clusters of crowded cells. There is a tendency for uniform nuclear enlargement in FC, whereas FA may show small or large nuclei.¹²⁴ These differences are often subtle, with much overlapping (Fig. 6.20).^3,68,125 Cells from a well-differentiated but clinically aggressive FC may not appear obviously atypical or enlarged in smears (Figs 6.23, 6.24B). Anisokaryosis per se is more a feature of non-neoplastic lesions such as NG and thyroiditis.⁴²

Most authors are content to use cytology to select cellular FNs for follow-up or surgical excision, and to leave a diagnosis of malignancy to histological assessment of capsular and vascular invasion. Reporting cytological atypia and any suspicion of malignancy, although not diagnostic, may be of some use in making the choice between follow-up and immediate surgical excision. The ultimate prognosis of microfollicular, solid or trabecular adenomas is uncertain and these should probably be excised anyway.

FC has been considered the second most common thyroid cancer, accounting for 10–20% of all thyroid malignancies.¹²⁶ The proportion of carcinoma in lesions designated as FN has been reported as ranging from 14% to 44% in previous series.^3,119 In a large series of operated cases from 1994 to 2002 from Bethesda,¹²⁷ an 18.2% rate of malignancy was found in cases in which a cytological diagnosis of ‘possible FN’ was given and 20.9% in cases with a definitive cytological categorization of ‘FN’.

Clinicians (and cytopathologists) are conventionally preoccupied with the inability of cytologically distinguishing FA from FC, leading to numerous ancillary investigations to help make this distinction. Computer assisted cell morphometry, ploidy analysis and determination of AgNORs in cytological smears have been tried with variable success.^61,128,129 Results of proton magnetic resonance spectroscopy have been encouraging.¹³⁰ However, the most promising technique for the future is probably immunocytochemical demonstration of molecular markers.¹³¹ Positive immunostaining with CD44v6 or galectin 3 (with a score of G2) in combination with FNA^54,55 have been noted to be useful in cases with indeterminate cytology.⁵⁶ Telomerase activity⁵⁸ and microarray analysis of cytologic samples⁵⁹ are other ancillary tests that may aid in distinguishing benign from malignant follicular lesions.

Interestingly, there are studies indicating that FC is gradually becoming a rare entity.^{18,22,24,132,133} Prevalence of malignancy in operated cases of FN¹¹⁸ was 31%, and 9% of these were follicular or Hurthle cell carcinomas. The incidence of FC was only 2% of thyroid cancers in LiVolsi’s series²⁴ and 1% at the University of Chicago Medical Center.²² Discussing the gradual demise of FC, De May²² opined that independent of the quality of any diagnostic test employed, statistically speaking, its predictive value would be affected by the prevalence of the disease in question. It stood to reason therefore that, if the prevalence of FC is low, the predictive value of cytology for FC would also tend to be low, although cytology is otherwise an excellent diagnostic modality.

Atypical adenomas show foci of extremely pleomorphic cells that cytologically simulate malignancy (Fig. 6.24A). These are designated as carcinoma only if there is capsular or vascular invasion. Nuclear pleomorphism is not a common feature of well-differentiated FC and is not considered a cytological criterion of malignancy. On the other hand, it is frequent in dyshormonogenetic goiter, treated GD, and following chemotherapy and radiotherapy.^42,85

In recent years, FV-PC has attracted much attention. Due to the presence of follicular groupings and colloid in these tumors (see Fig. 6.43), it constitutes a generous proportion of malignancies reported as FN.^21,22,118 However, a proportion of nuclei display typical cytological features of PC (described below in section on PC). In some series, high cytologic accuracy rates for FV-PC¹³⁴ with 93–94% sensitivity, specificity, positive and negative predictive values have been demonstrated using ultrafast PAP stain.¹³⁵

No cytological criteria clearly distinguish parathyroid from follicular thyroid neoplasms.¹³⁶ Agarwal et al.,¹³⁷performing US-guided FNA of 53 parathyroid adenomas, reported low sensitivity as a major limitation. Smears showed moderate cellularity with monomorphous, round to slightly oval cells predominantly arranged in loose two-dimensional clusters with occasional papillary fragments. The majority of them exhibited stippled nuclear chromatin and bare nuclei were seen in the background. There was no significant pleomorphism, mitotic activity, or prominent nucleoli. Parathyroid adenomas are usually not palpable but radiological examination is capable of locating most non-palpable parathyroid lesions. However, the occasional parathyroid adenoma may be intrathyroidal.¹³⁸ Cytohistological overlap in parathyroid and thyroid follicular lesions can be a problem at the time of frozen section evaluation, and intraoperative parathyroid hormone monitoring may be required. In parathyroid carcinomas, clinical, biochemical and radiological findings are usually characteristic. Cytology, combined with clinical, radiological and immunocytochemical findings can enhance diagnostic accuracy.¹³⁹ Tseleni-Balafouta¹⁴⁰ felt that due to overlapping morphological features, in order to avoid surgical mismanagement, the possibility of a parathyroid lesion should be stated clearly in cytology reports in all colloid-free cellular follicular lesions. Parathyroid incidentalomas comprise 0.4% of lesions in patients referred for suspected thyroid nodules, and parathyroid hormone analysis in FNA washouts has been found to be a diagnostic aid.¹⁴¹

FNs are often highly vascular and aspiration of blood may obscure neoplastic cells. It should be understood that excessively blood-stained smears are not necessarily related to poor technique but may signal the possibility of a neoplasm. Repeat needling using the fine needle capillary sampling technique with a 26 or 27-gauge needle after a gap of a week or two often yields diagnostic material.

Colloid in small follicles is often very dense and may be laminated, but the regular edge is unlike that of a psammoma body.