Keywords

Normal Cytology

The thyroid gland is an endocrine organ located anterior to the trachea and consists of two lobes connected at the midline by the isthmus. Depending on demographics and nutritional factors, the thyroid gland weighs between 14 and 18.5 g [1,2,3]. Pyramidal lobe, a normal component of the thyroid gland, can be seen in 15–75% [4]. Ultimobranchial bodies give rise to C cells (calcitonin-secreting cells), mostly seen in the mid-upper portion of thyroid lobes rather than peripheral areas and isthmus [4].

Fine needle aspiration (FNA) examination of the thyroid gland usually shows thyroid follicular cells that are smaller in size, arranged in a macrofollicular pattern, monolayer sheets, or often in mixed macro- and microfollicular patterns (Figs. 7.1 and 7.2). Follicular cells are small, close to the size of red blood cells, with round to oval nuclei, finely granular chromatin material, and smooth nuclear membrane. In addition, follicular cells can show pigment accumulation, such as hemosiderin. Colloid is usually present in the background, and appearance is variable in different cytology preparations and can either be watery or dense or thick. A watery colloid consists of a thin colloid layer and should be distinguished from the background serum. Color appearance is also variable and pink-orange or green in Papanicolaou and violet-blue with or without cracking artifact in Diff-Quik stain preparations. Calcium oxalate crystals can also be seen [5].

Fig. 7.1
A microscopic image displays a round patch and a cluster of prominently stained cells near to it. The cytoplasm is lightly stained.

Benign follicular cells with histiocytes (Papanicolaou stain × 400)

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Fig. 7.2
A microscopic image of minute scattered cells distributed all along the cytoplasm. A patch with deep staining is in the center of the image.

Cellular specimen with mixed microfollicular and macrofollicular cells with histiocytes. No cytologic atypia is seen. Overall features showed mix population of cells and consistent with cellular goiter (ThinPrep × 100)

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Other elements that are present include macrophages, cyst lining cells, lymphocytes, and Hurthle cells. Hurthle cells are oncocytic or oxyphilic cells with abundant granular cytoplasm and nucleoli that can be conspicuous [6]. They appear green or orange on the Papanicolaou stain and purple on Diff-Quik preparation.

Adequacy Criteria

Thyroid FNA cytology evaluation should have at least six groups of follicular cells with at least ten cells in each group, preferably on one slide. There are a few exceptions to this adequacy criterion in certain scenarios, such as abundant colloid (Figs. 7.3 and 7.4), lymphocytic thyroiditis, or nuclear atypia in follicular cells [7]. Follicular cells should be well preserved without any obscuring artifact.

Fig. 7.3
A microscopic image displays clusters of blood cells. They have an irregular, pointy shape. The cytoplasm is lightly stained and has a dotted appearance.

Thin colloid with background histiocytes (Diff-Quik × 100)

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Fig. 7.4
A microscopic image illustrates patchy structures in the central region with blood cells along the margin and the periphery of the cytoplasm.

Thick colloid and background red blood cells (Diff-Quik × 200)

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Fig. 7.5
A microscopic image has cells that are stained brightly and have a pointy appearance. The surrounding area does not have any cells and is lightly stained. A few clusters of pointy cells are at the 2 corners of the image.

Gel material used during the ultrasound procedure appears as amorphous material and should not be confused with colloid (ThinPrep × 400)

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Artifacts

Certain artifacts should be in consideration while evaluating thyroid FNA cytology specimens. (Table 7.1).

Table 7.1 Artifacts and contaminants
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Fig. 7.6
A microscopic image of fibrous tissue that is deeply stained with a few grainy nuclei scattered inside it. The cytoplasm is lightly stained.

Skeletal muscle fibers with striations can mimic colloid (Papanicolaou stain × 400)

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Graves’ Disease

Graves’ disease is an important cause of hyperthyroidism and is an autoimmune condition caused by circulating autoantibodies [21]. On ultrasound, it may show hypervascularization with an “inferno pattern” can be seen in color-doppler studies [22, 23]. Thyroid scintigraphy can show diffusely elevated uptake within the gland [24]. Overall, there is a diffuse enlargement of the gland which can cause hyperplastic thyroid follicles and peripherally scalloped colloid. Tall columnar cells with eosinophilic cytoplasm and scattered lymphocytes can be seen. However, nuclei are usually round, but nuclear clearing and papillary hyperplasia can pose diagnostic challenges. After radioactive iodine treatment, nuclear atypia and oncocytic metaplasia can be seen. The differential diagnosis includes papillary thyroid carcinoma; identifying the clinical history of Graves’ disease or RAI treatment is essential in such cases. Recognizing isolated nuclear features should be evaluated cautiously to avoid overinterpretation in the background of Graves’ disease.

Hashimoto’s (Chronic Lymphocytic) Thyroiditis

Hashimoto’s thyroiditis (HT) is an important cause of hypothyroidism and is autoimmune thyroiditis caused by antithyroid antibodies (such as antithyroid peroxidase and anti-thyroglobulin) [25]. Radiologically, it appears as a diffusely enlarged gland with various patterns (echogenicity) and can show variable changes [26]. Morphologic evaluation of the specimen shows oncocytic cells, a mixed population of lymphocytes, and plasma cells with a variable number of colloid materials (Fig. 7.8). Isolated nuclear cytologic atypia can be seen in HT, which may result in overinterpretation (Fig. 7.9) [27]. Oncocytic cells (Hurthle cells) can also show atypia which is more recognized as an endocrine type of atypia or degenerative atypia. The differential diagnosis includes lymphoma or papillary thyroid carcinoma. B-cell lymphoma is the most common primary thyroid non-Hodgkin lymphoma [28]. Lymphoma shows a monomorphic population of neoplastic lymphoid cells, while in HT, lymphocytes are polymorphous. Obtaining an additional pass for flow cytometry would be helpful in suspected cases of lymphoma. Papillary thyroid carcinoma should have typical nuclear features such as nuclear crowding, clearing, grooves, enlargement, and intranuclear pseudoinclusions.

Fig. 7.7
A microscopic image of cells that are dispersed along the cytoplasm. They are prominently stained and have a minute-round appearance. The cytoplasm is lightly stained.

Smearing artifact can produce a discohesive population of follicular cells which can resemble lymphocytes (Papanicolaou stain × 200)

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Fig. 7.8
A microscopic image reveals inflammatory cells that are distributed throughout the cytoplasm, appearing prominently and exhibiting deep staining, while the cytoplasm itself is lightly stained.

Lymphocytic thyroiditis (Papanicolaou stain × 200)

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Fig. 7.9
A microscopic image of a structure with a cluster of round, deeply stained cells. The cells are clustered inside the structure, while the surrounding area is lightly stained.

Lymphocytic thyroiditis with reactive nuclear changes (Papanicolaou stain × 400)

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Fig. 7.10
A microscopic image of structures with a cluster of cells that are prominent and slightly elongated. The deeply stained nuclei have a grainy appearance.

Histiocytic aggregate with a multinucleated giant cell and background inflammatory cells (ThinPrep × 400)

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Fig. 7.11
A microscopic image of the patchy appearance of a structure with dew lightly stained cells at the periphery.

Amyloid material with scattered spindle cells and multinucleated giant cells (Diff-Quik x 400)

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Fig. 7.12
A microscopic image illustrates an irregularly shaped patchy structure that has taken up the stain deeply. The surrounding region is lightly stained.

Amyloid material with scattered spindle cells and multinucleated giant cells (Papanicolaou stain × 400)

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Fig. 7.13
A microscopic image displays a wound that is stained and has a grainy appearance. Multiple single nuclei are scattered all along the cytoplasm.

Cyst lining cells with reactive changes and background histiocyte (ThinPrep × 400)

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Fig. 7.14
A microscopic image illustrates round and prominently stained nuclei. The structure has an irregular margin with a grainy appearance around the nuclei.

Sheet of follicular cells with granular pigment (brownish) (ThinPrep × 400)

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Figs. 7.15
A fine needle aspiration image with huge oblong peripheral cells. The cytoplasm and nuclei are prominent and fill the interstitial spaces. The central regions have a scaly appearance.

Prior FNA-related changes with histocytes, fibrosis, cholesterol cleft formation, and surrounding follicular cells with nuclear atypia (H&E × 40)

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De Quervain (Sub-Acute Granulomatous) Thyroiditis

De Quervain (sub-acute granulomatous) thyroiditis is a self-limiting inflammatory condition, mostly due to viral infection, usually presents with pain in the thyroid gland and hyperthyroidism. On imaging, color doppler studies usually show decreased flow with low uptake on radionucleotide scans [29]. Morphologic evaluation shows epithelioid histiocytes, multinucleated giant cells, and inflammatory cells (neutrophils in the early stage, then lymphocyte and plasma cells) (Fig. 7.10). The differential diagnosis includes other forms of thyroiditis, such as granulomatous thyroiditis, palpation thyroiditis, and papillary thyroid carcinoma. Granulomatous thyroiditis usually shows epithelioid histiocytes, and granuloma formation and infectious etiology (fungal and mycobacteria) should be ruled out. Palpation thyroiditis can have multinucleated giant cells but usually lack inflammatory infiltrate. Papillary thyroid carcinoma can come in differential diagnosis, especially in cellular cytology specimens with many multinucleated giant cells and epithelioid histiocytes, which can show nuclear clearing and overlapping. However, the lack of atypical nuclear features of PTC and the presence of inflammatory infiltrate with multinucleated giant cells and epithelioid histiocytes help to differentiate these two entities.

Riedel Thyroiditis

It is an uncommon fibrosing process involving the thyroid gland and usually presents as a firm neck mass [30]. There is a suggestion in the literature that Riedel thyroiditis is within the spectrum of IgG4-related diseases. [31] Morphologically, it shows fibrosis and inflammatory infiltrates, mostly plasma cells. Due to fibrosis, the cytology specimen is usually paucicellular. The differential diagnosis includes a paucicellular variant of anaplastic thyroid carcinoma (ATC) [14]. However, atypia is more in ATC cases, and PAX-8 stain would be helpful for further characterization.

Amyloid Goiter

Amyloid goiter can present as thyroid gland enlargement due to primary or secondary amyloidosis [32]. However, the most frequent setting in which amyloid is seen in the thyroid gland is medullary thyroid carcinoma. Cytology specimens are usually not cellular and mainly consist of amorphous amyloid material, which can resemble colloid material (Figs. 7.11 and 7.12). However, the presence of associated vasculature can be seen in amyloid [33]. Amyloid has been reported in parathyroid, which can mimic medullary thyroid carcinoma [34]. Medullary carcinoma is usually positive for neuroendocrine markers and calcitonin stains. Amyloid material is highlighted by Congo red stain and shows apple-green birefringence under polarized light [35]. Amyloid usually appears as bright yellow-green fluorescence when Thioflavin-T stain is examined under an immunofluorescent microscope. See Table 7.2.

Table 7.2 Benign and reactive conditions and differential diagnosis
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Thyroid Cystic Lesions

  1. 1.

    Thyroid nodule with degenerative cystic changes: Cystic thyroid nodules contain macrophages, watery colloid, mixed pattern follicular cells, and may be Hurthle cells. Cyst-lining cells can also be seen in flat sheets with slightly elongated nuclei but with distinct borders (Fig. 7.13) [36]. They can show Hurthloid, or spindled morphology, occasional nuclear grooves, degenerative change, rare mitosis, and reactive atypia can be seen and should be evaluated cautiously to avoid overinterpretation. Papillary thyroid carcinoma with cystic changes comes in the differential diagnosis, which should show classic nuclear features of PTC such as nuclear crowding, overlapping, nuclear grooves, pseudoinclusions, and papillary architecture.

  2. 2.

    Thyroglossal duct cyst: Thyroglossal duct cysts are usually in the midline neck and near the hyoid bone. FNA usually yield low cellularity specimen, mainly consisting of inflammatory cells, macrophages, and scant epithelial component/lining cells such as ciliated respiratory type epithelium or squamous epithelial cells. The Cyst wall may contain thyroid follicular cells. Carcinoma can arise in TGDC, which is mostly PTC [37]. The differential diagnosis of TGDC includes other cystic lesions such as dermoid cyst, which usually consists of adnexal structures, and brachial cleft cyst, primarily located in the lateral neck.

  3. 3.

    Parathyroid cyst: Parathyroid cysts are rare, mainly around the inferior parathyroid gland [38] and the left thyroid lobe. They can present in any age group, but most cases are noted between the third and sixth decade [39]. Cyst aspirate is usually clear but less cellular and consists of cyst contents and scant cuboidal epithelial cells. Cyst fluid analysis can be sent for parathyroid hormone levels analysis [40, 41]. The differential diagnosis includes thyroid follicular cells or thyroid cysts. If cell block is available, performing PTH or thyroid origin markers such as Thyroglobulin and TTF-1 would be helpful to differentiate these two entities. See Table 7.3.

Table 7.3 Cystic lesions
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Pigments, Crystals, and Calcifications

Pigments

Pigmented material (Fig. 7.14) can be seen in thyroid parenchymal cells, including hemosiderin, lipofuscin, due to tetracycline (minocycline, Black Thyroid) and melanin.

Hemosiderin

Hemosiderin pigment is a coarse granular golden yellow to brown pigment derived from erythrocyte breakdown. Hemosiderin deposition can be seen in macrophages and follicular cells primarily due to secondary causes such as cystic degenerative changes or prior FNA or biopsy procedures. An Iron stain (Prussian blue) highlights hemosiderin as blue cytoplasmic material.

Minocycline

Oxidative changes, deposition of derivative, or degradation products of minocycline can result in dark brown granular pigmentation of follicular cells [42, 43]. It is usually seen in a long-standing history of taking tetracycline (minocycline). Pigment may stain positive for Fontana-Masson and bleach with potassium permanganate but negative for iron stain [42, 44].

Lipofuscin

Cytoplasmic deposition of yellow to brown pigment is seen with aging and usually stain positive for lipofuscin stain and PAS-positive but negative for iron stain [44].

Melanin

Melanin is a fine granular brown-black cytoplasmic pigment. It is infrequently seen in the thyroid gland. However, rare cases of melanin-producing medullary thyroid carcinoma have been reported [45, 46]. Melanin stain with Fontana-Masson stain but negative for PAS.

Crystals

Calcium oxalate crystals are seen in the thyroid gland, specifically with colloid. They are birefringent under polarized light and can be found in all types of thyroid lesions and normal thyroid tissue [47]. They are more prevalent in goiter and adenomas [47]. the differential diagnosis includes calcifications. These crystals are rare in parathyroid tissue, and findings crystals by using polarized light microscopy would be helpful to differentiate thyroid tissue from parathyroid tissue in diagnostically challenging situations [48].

Calcifications

Dystrophic calcification: It is due to the deposition of calcified material in necrotic tissue or due to degenerative changes. They are usually coarse, dense, and irregular and do not show lamellation.

Psammoma bodies: They are basophilic and show concentric lamellation. They are often associated with papillary thyroid carcinoma. They can also be seen in benign conditions [49]. However, finding an isolated psammoma body without any atypical cells in thyroid aspirate may necessitate a comment on the presence of psammoma calcification with further correlation with clinical and imaging findings [50].

Infections

They are uncommon but can be seen due to bacterial, fungi, and mycobacterial infections.

Acute Suppurative Thyroiditis

It usually presents with fever, pain, and neck swelling due to bacterial infection, primarily by gram-positive bacteria (Staphylococcus aureus and Streptococcus species) [51]. Cytomorphologic exam may show inflammatory cells with a predominance of acute inflammation and necroinflammatory debris.

Granulomatous Thyroiditis

It can be infectious or non-infectious. Fungal (Aspergillus, Histoplasma, Candida spp., and others) [52, 53] and mycobacterial infections can cause inflammation, granuloma formation, and multinucleated giant cells reaction. Special stains and cultures are helpful for the identification and further characterization of the microorganism. Differential diagnosis includes non-infectious etiologies, including De Quervain (sub-acute) thyroiditis and sarcoidosis.

Thyroid Follicular Cells in Neck Region

Thyroid follicular cells can be seen in the central and lateral neck with associated differential diagnoses (Table 7.4).

Table 7.4 Thyroid follicular cells in the neck region
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FNA Induced Changes in Thyroid Gland and Differential Diagnosis

Fine needle aspiration (FNA) cytology helps in diagnosing thyroid lesions. However, the FNA procedure of the thyroid results in certain cytologic and histologic alterations, which are well described in the literature (Figs. 7.15). Early changes (less than 3 weeks) include the presence of hemorrhage, hemosiderin-laden macrophages, and granulation tissue. Morphologic changes that appear later include hemosiderin-laden macrophages, reactive cellular changes, fibrosis, and presence of myofibroblast cells, nuclear changes such as nuclear enlargement and clearing, metaplastic changes (squamous or oncocytic), infraction, cholesterol granuloma formation, vascular proliferation, and plumped endothelial cells [60,61,62,63]. In diagnostically challenging cases, cytomorphologic comparison with the prior FNA cytology may be helpful, if available.

Pitfalls

The presence of cytologic atypia and the above-described change in repeat thyroid FNA may pose diagnostic challenges. Nuclear atypia can stimulate features of papillary thyroid carcinoma. However, nuclear atypia due to post-FNA change is usually focal and lacks other features of PTC, such as intranuclear pseudoinclusions. The presence of spindle cells or myofibroblast-like cells, either due to reactive stromal changes or granulation tissue formation, can bring in a diagnosis of sarcoma or anaplastic thyroid carcinoma. Simultaneously, infarction due to prior procedure can stimulate necrosis which can be seen in thyroid malignancies.

Conclusion

To conclude, recognizing these cytologic changes and then the correlation with the clinical history (such as clinical/ imaging features and history of prior FNA procedure) is essential. Findings should be evaluated cautiously to avoid overinterpretation.