Fine Needle Aspiration Biopsy Cytology of Thyroid: A Diagnostic Approach Based on Pattern Recognition


Fine needle aspiration biopsy (FNAB) of the thyroid is currently the most accurate and cost-effective way of examining thyroid nodules (2009 American Thyroid Association guidelines). The number of thyroid FNAB has increased recently due to the number of thyroid nodules that are being discovered by ultrasound, which is used to supplement routine physical examinations. FNAB of the thyroid has made it possible to diagnose the vast majority of thyroid nodules greater than 10 mm, as well as smaller thyroid lesions and also cervical lymph nodes thought to be involved by metastatic thyroid carcinoma.

Ultrasound is used to screen thyroids for nodules, assess palpable nodules, and guide FNAB. Ultrasound for palpable nodules is useful if available, but FNAB by palpation has been a highly successful procedure for more than 50 years in Scandinavia and other countries. Every patient with a palpable or ultrasound-detected nodule larger than 1 cm is a candidate for FNAB, and if there is a history of radiotherapy or exposure to irradiation or family history of thyroid carcinoma, then ultrasound-directed FNAB of smaller lesions can be attempted.

At the same time, the incidence of thyroid carcinoma has been gradually increasing and is now the fourth commonest carcinoma in women. Follicular and papillary carcinomas arising from follicular epithelium have good prognoses with 10-year survival rates of greater than 90%. Medullary carcinomas arising from calcitonin-secreting C cells have a worse survival rate, similar or worse than aggressive variants of papillary carcinoma, including tall cell, columnar cell, diffuse sclerosing, solid, and the diffusely invading follicular variant, while the follicular cell–derived poorly differentiated carcinoma and anaplastic carcinoma have the worst prognoses.

Thyroid FNAB is an accurate diagnostic test for benign follicular nodules, follicular neoplasms, thyroiditis, and papillary carcinomas and most of these other carcinomas. Assessment of FNAB accuracy is difficult over time because the supposed “gold standard” of surgical pathology has shifted, with a decrease in follicular carcinoma and increase in papillary carcinoma and the “follicular variant of papillary carcinoma.”

Thyroid FNAB Accuracy

Thyroid FNAB has been recognized as the “most accurate and cost-effective method for evaluating thyroid nodules.”

FNAB has a positive predictive value (PPV) of greater than 90% overall for diagnosing specific thyroid lesions and a diagnostic accuracy for malignancy up to 98%. It is also highly accurate in diagnosing the various types of thyroiditis.

False negatives do occur because of undersampling of lesions, cystic papillary carcinomas, and lesions where there may be dual pathology, such as papillary carcinoma arising in a background of Hashimoto thyroiditis.

Traditionally there have been problems in distinguishing benign follicular or “adenomatous” nodules from “follicular neoplasms,” which include follicular adenomas and low-grade follicular carcinomas, but over time this has become less of a problem. Of course, the differential diagnosis (DD) of follicular adenoma and follicular carcinoma cannot be made on FNAB because this relies on demonstrating vascular invasion or capsular invasion at the margin of the nodule by histopathology. The clinical outcome for a benign follicular nodule and follicular adenoma is virtually identical, and most low-grade follicular carcinomas have a good prognosis.

Thyroid FNAB Technique

The technique and its possible but rare complications should be explained to the patient and the patient consented in an appropriate manner within the hospital or clinic. Warfarin should probably be replaced with heparin in patients medicated for anticoagulation. The thyroid is a very vascular organ, but use of fine needles carries a low risk of hemorrhage, although bleeding back into completely drained cysts does occur and can be minimized by placing firm pressure on the FNAB site after each pass.

Ultrasound of the thyroid can be used to confirm the palpable nodule and other possible target nodules. Lymph nodes should be carefully looked for because FNAB of these nodes at the same time as the thyroid can provide staging information. Although ultrasound is widely used, the cytopathologist can proceed to the FNAB using palpation alone for palpable nodules.

If the procedure is being performed under ultrasound for either a palpable or impalpable nodule, the cytopathologist can perform the FNAB closely with the radiologist or ultrasonographer, or the radiologist can perform the FNAB and an experienced cytotechnologist or cytopathologist can provide rapid on-site evaluation (ROSE) to evaluate adequacy and triage the specimen for possible ancillary studies such as flow cytometry. ROSE is cost-effective, reducing the number of inadequate specimens and the need for repeat FNAB, which involves costly repeat ultrasound and FNAB, as well as providing best practice and cost-effective use of the specimen and minimizing patient discomfort.

The patient can be in a reclining chair or on a bed, but the neck needs to be extended over a low pillow to expose the thyroid. This should be done carefully in the elderly and may be uncomfortable in those with any arthritic neck condition. The patient should not be left supine in this position for long periods of time.

If there are multiple nodules, then those nodules showing any atypical ultrasound features should be selected, or multiple nodules can undergo FNB or a nodule in each lobe can be sampled.

Fine needles of 23, 25, or 27 gauge should be used. Because nodules in the thyroid frequently have cystic degeneration, use of a needle by itself to perform the FNAB may lead to cyst fluid cascading out of the needle. A syringe can be attached to the needle for the FNAB, or an aspiration syringe holder or “gun” can be used, which frees up the other hand to palpate and “hold” the nodule with the fingertips. Alternatively, if the patient is supine, a needle attached to a syringe with the plunger removed can be used to aspirate cysts.

An attempt should be made if cyst fluid flashes immediately back into the needle hub to drain the cyst completely. This can be quite dramatic when viewed under ultrasound or felt under the palpating fingers. Hemorrhage can occur virtually immediately back into the cyst, so firm pressure should be applied to the cyst bed, after drainage. The operator should record on the request form if the cystic lesion is completely drained.

If there is a residual nodule, either by palpation or seen on ultrasound, a needle by itself can be used to rebiopsy the lesion. This technique, which can also be used for the first pass if the ultrasound shows a definite solid nodule, allows fine control of the needle and facilitates the required rapid “woodpecker” passaging of the needle deeply into the nodule.

At least two and up to four passes or needlings of the nodule are recommended to increase accuracy, first with the syringe holder to drain cysts and then with the needle only, that is, the nonaspiration technique of any residual lesion.

When the needle enters the vascular thyroid, blood appears rapidly in the hub. The needle should be withdrawn after one or two more rapid jabs. Unless the hub fills immediately with brown cyst fluid followed by a surge of easily aspirated cyst contents, the needle should not be kept in the nodule. Filling the syringe with 1 or 2 mL of bright red blood produces poor bloody smears with diluted diagnostic tissue, and invariably dwelling for more than 5 to 10 seconds in the thyroid while ultrasound pictures are taken to “prove the needle was in the lesion” leads to fibrin clotting, especially on the second and subsequent passes.

The essence of the good thyroid FNAB is to use a 25- or 27-gauge needle to rapidly stab the nodule, agitating the needle into and out of the nodule rapidly over a 5- to 10-second period, and remove it immediately after blood enters the hub. Aspiration should NOT be used during this “fine needle biopsy.” The exceptions are the cyst that should be drained and the colloid nodule that will resist even aspiration of its tenacious and diagnostic thin colloid.

Both air-dried Giemsa-stained and alcohol-fixed Papanicolaou (Pap)-stained slides should be used for all cases, recognizing that some institutions will have a long tradition of only using one of these stains or a variant such as the H&E stain.

Cell blocks are useful in some circumstances for ancillary studies, such as where medullary carcinoma or metastasis is suspected clinically or at ROSE. It is up to the individual cytopathologist to decide if a cell block should be made.

Liquid-based preparations offer the advantages that all the material is placed in a fluid medium, so direct smears fixed in alcohol for Pap stains will never show “air-drying artifact,” but it is not possible to prepare air-dried Giemsa stains with this technique and, importantly, most of the patterns that provide so much diagnostic information and most of the thin colloid are lost. The notable advantage of the liquid-based preparations is that the whole case is reduced to one slide, and sometimes it helps to reduce inadequacy rates when the smears are pauci-cellular. However, noncytology staff can be trained to achieve good-quality direct smears, and ROSE reduces the inadequacy rates of thyroid FNAB and provides both alcohol-fixed Pap-stained and air-dried, Giemsa-stained slides in a cost-effective manner. The method of air-drying slides at the FNAB and then rehydrating before Pap staining has been advocated.

Thyroid FNAB: Criteria for Adequate Smear

The Bethesda System for Reporting Thyroid Cytopathology (TBS) defines thyroid FNAB adequacy. It requires a minimum of six tissue fragments or sheets. Each sheet has 10 to 20 cells so that the architecture of the sheets can be seen, and a colloid background may be helpful. A single, large tissue fragment of multiple follicles and intervening stroma can suffice.

However, in a colloid nodule with a large amount of thin colloid there may be minimal or no follicular epithelium, but the distinctive appearance of the slide seen macroscopically or when stained is regarded as sufficient by TBS. When correlated with the clinical and imaging findings, these cases should be signed out as a “colloid nodule.” Similarly, with chronic lymphocytic thyroiditis there is no requirement to have follicular sheets in the presence of reactive lymphoid material. If there is any atypia such as intranuclear pseudoinclusions or marked nuclear atypia in any technically insufficient smear, then this atypia should be reported.

Cystic degeneration is common in benign follicular nodules and follicular neoplasms and can occur in papillary carcinomas of the thyroid and in metastases. It is contentious whether follicular epithelium is necessary in the cystic background of siderophages, debris, old blood, and thin colloidal proteinaceous background to make the FNAB adequate. If the lesion is fully drained at ultrasound and the radiologist sees no atypical features on ultrasound, or there is no residual nodule to palpate, it is reasonable to accept a “cyst contents” diagnosis, recording that there was no epithelium. But under the current TBS, cyst contents without follicular epithelium in the absence of ultrasound or clinical information that the cyst has been drained completely is regarded as “nondiagnostic” or “insufficient.”

Thyroid FNAB Request Form

It is essential that the referring clinician provide the standard demographic information, any family history related to thyroid pathology, and history of exposure to radiation and antithyroid treatment to the cytopathologist. Each thyroid nodule undergoing FNAB should be documented as to the size, specific site (lobe and position in the lobe, “upper” or “lower,” etc.), and imaging findings.

Thyroid FNAB Diagnostic Terminologies

In October 2007, the U.S. National Cancer Institute and Papanicolaou Society of Cytopathology sponsored a conference to determine the use of FNAB in the management of thyroid nodules and to standardize the diagnostic terminology, which led to the publication of a classification system and an atlas. The classification included six distinct, well-defined cytomorphologic categories that matched the probability of malignancy at surgery to the category and the corresponding clinical management recommendations ( Tables 4-1 and 4-2 ). It introduced standardized nomenclature and diagnostic criteria and set phrases to be used in reporting diagnoses with the hope to improve intrainstitutional and interinstitutional reproducibility of thyroid FNAB reporting and to gain wider clinical acceptance of thyroid FNAB. It provided relatively unambiguous, clear, and clinically relevant diagnostic categories, linking FNAB diagnosis and patient management. TBS does not advocate using category numbers in reports but rather establishes set diagnostic phrases. Potentially it will replace multiple reporting systems, but it has attracted considerable criticism, as well as support.

Table 4-1
Diagnostic Categories of the Bethesda System for Reporting Thyroid Cytopathology
Diagnostic Category Risk of Malignancy (%)
  • I.

    Nondiagnostic

  • II.

    Benign

0-3
  • III.

    Atypia of undetermined significance

5-15
  • IV.

    Follicular neoplasm/suspicious for follicular neoplasm

15-30
  • V.

    Suspicious for malignancy

60-75
  • VI.

    Malignant

97-99

Table 4-2
Clinical Management of Different Categories of the Bethesda System for Reporting Thyroid Cytopathology
Diagnostic Category Clinical Management
  • I.

    Nondiagnostic or unsatisfactory

Repeat FNA (>3 mo, 60% repeat FNA diagnostic)
  • II.

    Benign

Clinical follow-up
  • III.

    AUS/FLUS

Repeat FNA (repeat FNA 20%-25% still AUS)
  • IV.

    FN/SFN

Surgical lobectomy
  • V.

    Suspicious for malignancy

Near-total thyroidectomy or lobectomy
  • VI.

    Malignant

Near-total thyroidectomy
FNA, fine needle aspiration; AUS/FLUS, follicular lesion of undetermined significance; FN/SFN, follicular neoplasm/suspicious for a follicular neoplasm.

The TBS has three categories for indeterminate FNAB because each category has its own risk of malignancy. “Atypia of undetermined significance” (AUS) or “follicular lesion of undetermined significance” (FLUS) are cases that are not diagnostic of a specific lesion and have features that cause some concern but have a low risk of malignancy in the range 5% to 15%. Cases that raise suspicion of a follicular neoplasm, that is, follicular adenoma or follicular carcinoma with capsular or vascular invasion, have a more substantial risk of malignancy, 15% to 30% in the literature, while those that are suspicious of carcinoma, such as papillary carcinoma including the follicular variant of papillary carcinoma, have a high risk of malignancy in subsequent surgery of 60% to 75%.

The AUS/FLUS category is a heterogeneous group, supposedly representing less than 7% of all the FNAB, but more recent publications have shown that the rate of AUS/FLUS can range to greater than 20%. TBS has been criticized for introducing this potential “wastebasket” of cases, which potentially could encourage a lack of rigor in the diagnostic process. TBS sensibly only allows eight specific smear findings for this diagnosis ( Table 4-3 ), but there have been many publications on the institutional impact of the classification system on inadequate rates and AUS rates, using both direct smears and liquid-based preparations. AUS rates have been reported as between 3% and 18% by an analysis of eight papers, and an AUS-to-malignancy ratio at surgery has been proposed as a quality assessment measure, suggesting that a rate of 3:1, AUS to surgically proven malignancy, is appropriate. If the AUS-to-malignancy ratio is greater than 3, then either AUS is being overdiagnosed or malignancy is underdiagnosed.

Table 4-3
Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance: Eight Scenarios in the Bethesda Atlas
  • 1.

    Predominance of microfollicles in scant slide/scant colloid

  • 2.

    Predominance of oncocytic cells in scant slide/scant colloid

  • 3.

    Follicular cell atypia but air-drying artifact

  • 4.

    Cellular smear with predominance of oncocytic cells but in clinical setting of multinodular goiter or Hashimoto thyroiditis

  • 5.

    Focal nuclear features of papillary carcinoma in smears typical of Hashimoto thyroiditis

  • 6.

    Atypical cyst lining epithelium with grooves and occasional inclusions, in cystic background with follicular cells

  • 7.

    Focal follicular atypia with nuclear enlargement and nucleoli, plus history of radioactive iodine, radiotherapy, or cystic lesion

  • 8.

    Atypical lymphoid infiltrate and no flow cytometry available

Table 4-4
Comparing the Three Systems for Thyroid Reporting: The Bethesda System, 2014 Italian and British
From Nardi F, Basolo F, Crescenzi A, Fadda G, Frasoldati A, Orlandi F, et al. Italian consensus for the classification and reporting of thyroid cytology. J Endocrinol Invest . 2014;37(6):593-599 and Pagni F, Prada M, Goffredo P, Isimbaldi G, Crippa S, Di Bella C, et al. “Indeterminate for malignancy” (Tir3/Thy3 in the Italian and British systems for classification) thyroid fine needle aspiration (FNA) cytology reporting: morphological criteria and clinical impact. Cytopathology . 2014;25(3):170-176.
The Bethesda System for Reporting Thyroid Cytopathology The British System to Report Thyroid Cytology The Italian System to Report Thyroid FNA
Nondiagnostic or unsatisfactory Thy1. Nondiagnostic TIR1. Nondiagnostic
Benign Thy2. Nonneoplastic TIR2. Negative for malignant cells
Atypia of undetermined significance or follicular lesion of undetermined significance Thy3. Neoplasm possible TIR3. Inconclusive/indeterminate (follicular proliferation)
Follicular neoplasm or suspicious for a follicular neoplasm
Suspicious for malignancy Thy4. Suspicious for malignancy TIR4. Probably malignant
Malignant Thy5. Malignant TIR5. Positive for malignant cells

Recently one of the original authors of the Bethesda classification suggested that the classification should be left as it is for a further period of time, to allow further assessment, before any change is promoted. On the other hand, subclassification of the AUS/FLUS category into those with scant material showing a predominantly microfollicular pattern, prominent oncocytic/Hurthle cells or suboptimal smearing and staining, and those with nuclear atypia suggestive of papillary carcinoma does show that nuclear atypia carries a greater risk of subsequent carcinoma. It is not recommended at present to use “FLUS” for the former and “AUS” for the latter. Applying molecular tests, such as BRAF, which has near 100% specificity for papillary carcinoma, to the AUS/FLUS case with nuclear atypia rather than all AUS/FLUS cases may be cost-effective in determining which patients go to thyroidectomy. However, subclassification of the AUS/FLUS category at the present time has not led to changes in management, and repeat FNAB remains the least expensive and so far proven technique to clarify an initial AUS/FLUS diagnosis. Approximately 50% of AUS diagnoses will show a follicular neoplasm or more significant lesion at repeat FNAB or surgical biopsy.

It should be emphasized that the six categories should never be reported purely as a category number as the diagnosis in a report, but rather the standard phrases embodied in the classification should be used in the FNAB report, to foster clear communication between the clinician and cytopathologist. The categories can be recorded by number for quality assurance statistical analysis.

Ancillary Studies of Thyroid FNAB

Although the Bethesda System for Reporting Thyroid Cytopathology is widely accepted in the United States, the indeterminate cytologic diagnoses pose problems for both pathologists and clinicians. This has led to the development of molecular markers to attempt to improve FNAB diagnosis and tumor prognostication. Over the past decade, a number of molecular markers have emerged as possible diagnostic tools for solving this challenge.

Protein-Based Analysis (Immunohistochemistry)

Several markers have been studied in order to differentiate non-neoplastic lesions from neoplastic thyroid nodules.

Cytokeratin 19 has been shown to be strongly positive in papillary thyroid carcinoma including the follicular variant. Although reactivity to CK19 is reported to be more than 90% for papillary carcinoma, it can be positive in other non-neoplastic lesions and benign neoplasms of the thyroid. Currently, the use of CK19 as a sole marker for papillary carcinoma on FNAB is discouraged.

Galectin 3 is a β-galactosil-binding protein involved in regulating the cell cycle of thyroid cell transformation. It has been shown that galectin 3 is strongly expressed in papillary carcinoma. However, up to 60% of benign thyroid nodules are also positive for galectin 3. Furthermore, it is still controversial as to whether galectin 3 can differentiate between Hurthle cell adenoma and Hurthle cell carcinoma. Thus its use in thyroid FNAB is not widely accepted.

HMBE-1 recognizes an unknown epitope present on the microvilli of mesothelial cells. Follicular carcinoma (40%) and papillary thyroid carcinoma (85% to 100%) show diffuse positivity, while most benign lesions only show focal positivity. The caveat is that negative HBME-1 stain does not rule out carcinoma.

Mutation Analysis

Mutation of the BRAF gene is the most common genetic alteration in papillary thyroid carcinoma with a positive predictive value close to 99%. It is frequently positive in classic papillary thyroid carcinoma and the tall cell variant of papillary thyroid carcinoma, which are usually readily diagnosed by FNAB but is positive less frequently in the follicular variant of papillary carcinoma with a sensitivity as low as 25%. The follicular variant is the most difficult to diagnose on FNAB. A number of studies have shown that many BRAF -positive indeterminate cytology cases are papillary carcinomas on surgical follow-up and that BRAF performed on FNAB cell blocks is helpful in establishing the definitive diagnosis of malignancy. A recent study shows that refined morphologic criteria and BRAF mutational analysis enhance the assessment of preoperative risks for papillary carcinoma among “suspicious for papillary thyroid carcinoma” cases. When positive, it suggests a more aggressive clinical course with more frequent regional lymph node metastases, distant metastases, and recurrent disease. Thus surgeons may include a central neck or level IV lymph node dissection at the time of the total thyroidectomy. Rossi and colleagues recently demonstrated that specific cell features that include plump cells and sickle-shaped malignant nuclei are highly associated with the BRAF mutation.

The PAX8–PPAR-γ fusion protein is found in 23% to 63% of follicular thyroid carcinomas and results in overactive cell proliferation and differentiation. The diagnostic value of this mutation as a stand-alone test to differentiate benign from malignant indeterminate FNAB is limited by its expression in 2% to 13% of benign thyroid lesions.

Since there is no single molecular test that can solve the problematic issue of indeterminate cytology, testing FNAB samples for a panel of mutations offers greater predictive power for malignancy. A recent study explored the diagnostic utility of a panel of mutations including BRAF, RAS, RET/PTC, and PAX8-PPAR-γ. The presence of any mutation was a strong predictor of cancer, as 97% of mutation-positive thyroid nodules had surgically proven malignancy.

Gene Expression Analysis

Gene expression profiling, high throughput, and computational analyses have provided new methods to identify potential target genes or gene panels to differentiate benign from malignant thyroid lesions. FNAB of thyroid provides excellent material for these tests.

The Veracyte Afirma gene classifier is a multigene expression classifier that can be used to assess thyroid nodules of indeterminate nature that have been classified usually as atypia of uncertain significance (AUS) or follicular lesion of uncertain significance (FLUS), particularly if the repeat FNAB renders the same AUS diagnosis. The test is also useful for the diagnosis “suspicious of follicular neoplasm.” A recent study suggests the test is of less use in cases with oncocytic differentiation. It uses mRNA extracted from FNAB material to measure expression levels of 167 genes to identify the signature of gene expression in a multidimensional algorithm, with the aim of ruling out carcinoma in thyroid nodules where there is indeterminate cytology, because the test has a high negative predictive value. The initial trial performed on 24 indeterminate FNAB samples showed that the sensitivity and specificity of the test for malignant nodules are 85% and 40%, respectively. One of the major flaws of the study was the small number of indeterminate FNAB samples. A more recent study applied the Veracyte Afirma gene classifier to 265 indeterminate FNAB cases. Of the 85 malignant nodules on final histology, the gene classifier predicted 92% correctly with a specificity of 52%. However, there are two major problems with this test influencing its wider use: firstly, the specificity is low, and the negative predictive value in a patient with AUS/FLUS due to nuclear atypia still does not totally exclude malignancy ; and secondly, the cost is high, projected to be 3200 USD for the test alone.

miRNA Analysis

Micro ribonucleic acid (miRNA) has also been shown to function as a tumor suppressor or oncogene in cancerous cells and is useful for cancer prognostication and classification. Several studies have investigated the use of miRNA to differentiate between benign and malignant thyroid nodules on histology, but few studies have applied the test to indeterminate FNAB. One study by Nikiforova and colleagues studied the utility of seven miRNA in thyroid FNAB cases in order to differentiate benign from malignant nodules. Only eight FNAB samples were indeterminate, and with the cutoff point at one of seven miRNAs upregulated, the sensitivity and specificity of the test were 88% and 94%, respectively. A recent study analyzed miRNA in 101 indeterminate thyroid FNAB samples. The reported sensitivity and specificity were 100% and 86%. This study remains the largest miRNA analysis of indeterminate thyroid nodules to date and provides useful information on feasibility and accuracy of miRNA prediction models for indeterminate thyroid lesions. The recently commercially available but still expensive miRinform test confirms malignancy in indeterminate cases with a positive predictive value of around 87%. Potentially this will reduce the number of cases where a total thyroidectomy has to be carried out after hemithyroidectomy has been performed for an indeterminate cytology case.

Targeted next-generation sequencing panel (ThyroSeq)

Next-generation sequencing (NGS) technology allows sequencing of multiple selected genomic regions and up to the whole genome. Since it is an amplification-based approach, it only requires a small amount of DNA. This is of particular interest for FNAB and small tissue biopsy samples. In a recent study by Nikiforov and colleagues, NGS was applied to 143 consecutive “suspicious for follicular neoplasm” thyroid FNAB cases, and “ThyroSeq” shows a 90% sensitivity, 93% specificity, and 83% positive predicative value in predicting malignancy.

Although the previously mentioned studies have shown promising results, there are several problematic issues. The prevalence of these mutations is highly variable, for example, BRAF mutation was found in 45% to 69% of papillary carcinomas from the United States and western countries, but over 80% in some of the Asian countries, such as South Korea. Thus it may be more meaningful to perform BRAF analysis in a BRAF -prevalent population. Also, the methodologies of these tests are highly variable and lack standardization, while the choice of a method is largely dictated by the type of samples, such as freshly collected FNAB or formalin-fixed cell block materials. Using an inappropriate method can result in loss of specificity of the test. Finally, most of these studies apply molecular tests on indeterminate thyroid FNAB, where the interobserver variability in classifying these so-called indeterminate FNAB is relatively high. In summary, molecular diagnostic markers, when used properly, combined with refined cytomorphologic criteria show promise in improving the management of indeterminate FNAB cases but are currently not used routinely in most medical centers.

Smear Patterns: Specific Lesions and Their Cytopathological Criteria

When assessing thyroid FNAB smears, an experienced cytopathologist or cytotechnologist can readily make the diagnosis of most cases on the smear pattern using scanning powers, confirming the nuclear features in the epithelial tissue fragments and the nature of the dispersed cells on high power, repeating this process, low to high power, until the findings correlate. If the high power is discrepant, for instance, if plentiful nuclear pseudoinclusions are found in a follicular pattern with colloid, the low power has to be reassessed.

Some disease processes and tumors can present with different patterns. Chronic lymphocytic thyroiditis, which with supporting clinical evidence may be called Hashimoto thyroiditis, can present with multinucleated giant cells and be regarded as “granulomatous” but lacks prominent epithelioid granulomas, while it can also present with mainly lymphoid material and scant epithelium and be regarded as a lymphoid inflammatory pattern. There is a spectrum of lymphocytic thyroiditis and Hashimoto thyroiditis with the cutoff based on clinical and serologic findings and the cytology. The cytoplasm of hyalinizing trabecular tumors can be poorly defined, and it may show syncytial sheets of tumor cells, while on the other hand, it can show more defined cytoplasm and more trabecular tissue fragments. For practical purposes, we placed it in Pattern 7 with papillary carcinoma because the presence of often plentiful nuclear pseudoinclusions leads to a DD with papillary carcinoma.

The term oncocytic rather than oxyphilic or Hurthle or Askanazy to describe the cells in Pattern 4-6 is controversial but preferred to these other historical terms, which have largely been used in histopathology.

“Poorly differentiated carcinoma” is a poorly defined lesion but in most series reflects a primary thyroid carcinoma without papillary or follicular or other differentiation and lacks the severe anaplasia of anaplastic carcinoma, but the distinction in each case reflects a spectrum of dysplasia. Metastases to thyroid can produce a number of different patterns and are included in the DD of high-grade primary carcinomas.

The eight thyroid FNAB smear patterns are presented in Table 4-5 .

Table 4-5
Eight Smear Patterns for Fine Needle Aspiration Biopsy Assessment
  • 1.

    Inflammatory cells predominate

    • i.

      Suppurative with or without organisms

      • a.

        Bacterial

      • b.

        Fungal

    • ii.

      Granulomatous

      • a.

        Subacute granulomatous thyroiditis (De Quervain)

      • b.

        Mycobacterial thyroiditis

    • iii.

      Lymphoid

      • a.

        Chronic lymphocytic thyroiditis/Hashimoto thyroiditis

      • b.

        Mucosa-associated lymphoid tissue (MALT) lymphoma

      • c.

        Monotonous small to intermediate lymphoid cells: small cell lymphomas

      • d.

        Monotonous intermediate/large lymphoid cells: intermediate to large cell lymphomas

  • 2.

    Cystic background with evidence of old hemorrhage

    • i.

      With colloid and follicular tissue fragments

    • ii.

      With papillary tissue fragments

  • 3.

    Predominant thin colloid with scant follicular epithelial sheets

  • 4.

    Follicular tissue fragments and larger fragments in which fibrovascular strands separate follicles of varying size, in a background of usually abundant thin colloid

    • i.

      Normal thyroid

    • ii.

      Benign follicular nodule

    • iii.

      Hyperplastic thyroid disease (Graves disease)

  • 5.

    Epithelial tissue fragments predominantly microfollicles and scant colloid

    • i.

      Follicular neoplasm: follicular adenoma or low-grade follicular carcinoma with capsular or vascular invasion

    • ii.

      Follicular variant of papillary carcinoma

    • iii.

      Parathyroid tumor

  • 6.

    Epithelial tissue fragments with granular cytoplasm and scant or no colloid

    • i.

      Oncocytic neoplasm: Hurthle cell adenoma or carcinoma

    • ii.

      Oncocytic variant of follicular carcinoma

    • iii.

      Oncocytic variant of papillary carcinoma

    • iv.

      Medullary carcinoma

    • v.

      Metastastic carcinoma including renal cell carcinoma

  • 7.

    Epithelial tissue fragments with well-defined cytoplasm, nuclear grooves and pseudoinclusions and variable papillary tissue fragments and variable colloid

    • i.

      Papillary carcinoma

    • ii.

      Hyalinizing trabecular tumor

  • 8.

    Predominantly single cells with epithelioid or spindle cells

    • i.

      Medullary thyroid carcinoma

    • ii.

      Poorly differentiated (“insular”) carcinoma of thyroid

    • iii.

      Anaplastic carcinoma of thyroid

    • iv.

      Metastatic tumors including melanoma and lobular breast carcinoma

In terms of the Bethesda System of reporting, in which the diagnoses are based on both low-power and high-power assessment, smear patterns 1, 2, 3, and 4 broadly correlate with the “benign” category, while smear pattern 5 correlates with “suspicious of a follicular neoplasm” or “suspicious of carcinoma,” and smear pattern 6, 7, and 8 correlate with “suspicious of carcinoma” or “carcinoma.” The smear pattern is the initial assessment of the FNAB material, and the DD of each smear requires high-power assessment to decide which of the differential diagnoses of that pattern is present.

Plate 4-1
Pattern 1 Inflammatory Cells Predominate

Plate 4-1A, Pattern 1: Demonstrated by the granulomatous pattern of subacute granulomatous thyroiditis: plentiful large multinucleated giant cells and lymphocytes and histiocytes in a background of debris, relatively degenerate sheets of follicular cells, and fragments of thick colloid, some partially engulfed by giant cells. (Giemsa ×4)

Plate 4-1B, Pattern 1: Sheets of mildly degenerate follicular cells are seen top center with thick colloid top right, with plentiful lymphocytes and the larger histiocytes and multinucleated histiocytes in a background that may have some debris. Note the irregular arrangement of the nuclei in the multinucleated histiocytes, compared with the regular arrangement of nuclei seen in intact follicles.

Plate 4-1C, Pattern 1: Demonstrated by the granulomatous pattern of subacute granulomatous thyroiditis: scattered multinucleated giant cells in a “dirty” background of lymphocytes, degenerate cells, and debris. (Pap ×10)

Plate 4-1 Table
Pattern 1: Inflammatory Cells Predominate.
Entities in the Differential Diagnosis

  • i.

    Suppurative with or without organism

    • a.

      Bacterial

    • b.

      Fungal

  • ii.

    Granulomatous

    • a.

      Subacute granulomatous thyroiditis (De Quervain)

    • b.

      Mycobacterial thyroiditis

  • iii.

    Lymphoid

    • a.

      Chronic lymphocytic thyroiditis/Hashimoto thyroiditis

    • b.

      Mucosa-associated lymphoid tissue (MALT) lymphoma

    • c.

      Monotonous small to intermediate lymphoid cells: small cell lymphomas

    • d.

      Monotonous intermediate to large lymphoid cells: intermediate to large cell lymphomas

Plate 4-2
Pattern 2 Cystic Background With Evidence of Old Hemorrhage

Plate 4-2A, Pattern 2: Scattered follicular sheets showing some evidence of oncocytic change in a thin colloidal background with siderophages. (Giemsa ×10)

Plate 4-2B, Pattern 2: Occasional follicular or oncocytic sheets, with their greater amount of granular cytoplasm, along with histiocytes, siderophages, and multinucleated histiocytes, in a thin colloidal or cystic background. Note the siderophages can aggregate.

Plate 4-2C, Pattern 2: Occasional small follicular sheets with siderophages and histiocytes in a thin colloidal background. (Pap ×10)

Plate 4-2 Table
Pattern 2: Cystic Background With Evidence of Old Hemorrhage
Entities in the Differential Diagnosis

  • i.

    With colloid and follicular tissue fragments

  • ii.

    With papillary tissue fragments

Plate 4-3
Pattern 3 Predominant Thin Colloid With Scant Follicular Epithelial Sheets: Colloid Nodule

Plate 4-3A, Pattern 3: Very scattered sheets of follicular epithelium in a background of abundant thin colloid from a colloid nodule. (Giemsa ×2)

Plate 4-3B, Pattern 3: Occasional follicular sheets with well-ordered nuclei and pale cytoplasm in a background of copious thin colloid showing cracking.

Plate 4-3C, Pattern 3: Occasional small sheets of follicular epithelium in a background of abundant thin colloid showing fine bubbling. (Pap ×10)

Plate 4-3Table
Pattern 3: Predominant Colloid With Scant Follicular Epithelial Sheets: Colloid Nodule
Entities in the Differential Diagnosis

  • i.

    Colloid nodule

Plate 4-4
Pattern 4 Follicular Tissue Fragments and Larger Tissue Fragments in Which Fibrovascular Strands Separate Follicles of Varying Size in a Background of Usually Abundant Thin Colloid

Plate 4-4A, Pattern 4: Benign follicular nodule showing large tissue fragments consisting of follicles of varying size including macrofollicles separated by stroma in a background of abundant thin colloid showing cracking. (Giemsa ×2)

Plate 4-4B, Pattern 4: Tissue fragment of follicles separated by stroma at bottom right, sheets of follicular epithelium from macrofollicles, and intact follicles are present in a background of usually abundant thin colloid showing cracking.

Plate 4-4C, Pattern 4: Large tissue fragment consisting of follicles of varying size separated by stroma and occasionally containing thick colloid, in a thin colloid background with some debris and fibrin clotting from a benign follicular nodule. (Pap ×4)

Plate 4-4 Table
Pattern 4: Follicular Tissue Fragments and Larger Tissue Fragments in Which Fibrovascular Strands Separate Follicles of Varying Size, in a Background of Usually Abundant Thin Colloid
Entities in the Differential Diagnosis

  • i.

    Normal thyroid

  • ii.

    Benign follicular nodule

  • iii.

    Hyperplastic thyroid disease (Graves Disease)

Plate 4-5
Pattern 5 Epithelial Tissue Fragments Predominantly Microfollicles and Scant Colloid

Plate 4-5A, Pattern 5: Follicular neoplasm (follicular adenoma confirmed on surgical pathology) showing high cellularity, large tissue fragments, and sheets of cells showing a microfollicular architecture in a relatively clean background. (Giemsa ×2)

Plate 4-5B, Pattern 5: Cellularity is high, with plentiful microfollicles and sheets of follicular epithelium showing microfollicles in a background which tends to be clean with minimal thin colloid.

Plate 4-5C, Pattern 5: Follicular neoplasm showing small tissue fragments with a microfollicular pattern in a background of almost no thin colloid. (Pap ×4)

Plate 4-5 Table
Pattern 5: Epithelial Tissue Fragments Predominantly Microfollicles and Scant Colloid
Entities in the Differential Diagnosis

  • i.

    Follicular neoplasm: follicular adenoma or low grade follicular carcinoma with capsular or vascular invasion

  • ii.

    Follicular variant of papillary carcinoma

  • iii.

    Parathyroid tumor

Plate 4-6
Pattern 6 Epithelial Tissue Fragments With Granular Cytoplasm and Scant or No Colloid

Plate 4-6A, Pattern 6: Oncocytic (Hurthle cell) neoplasm showing moderate cellularity, pattern of small tissue fragments showing in some a microfollicular architecture in a background of scant thin colloid. (Giemsa ×5)

Plate 4-6B, Pattern 6: Oncocytic cells in sheets and as single cells with well-defined granular cytoplasm, with occasional binucleates, in a clean background.

Plate 4-6C, Pattern 6: Oncocytic neoplasm Showing high cellularity with small sheets of oncocytic cells with granular cytoplasm. (Pap x10)

Plate 4-6 Table
Pattern 6: Epithelial Tissue Fragments With Granular Cytoplasm and Scant or No Colloid.
Entities in the Differential Diagnosis

  • i.

    Oncocytic neoplasm: Hurthle cell adenoma or carcinoma

  • ii.

    Oncocytic variant of follicular carcinoma

  • iii.

    Oncocytic variant of papillary carcinoma

  • iv.

    Medullary carcinoma.

  • v.

    Metastatic carcinoma including renal cell carcinoma

Plate 4-7
Pattern 7 Epithelial Tissue Fragments With Well-Defined Cytoplasm, Nuclear Grooves and Pseudoinclusions, and Variable Papillary Tissue Fragments and Variable Colloid

Plate 4-7A, Pattern 7: Papillary carcinoma showing highly cellular smear showing sheets and tissue fragments of epithelium with denser cytoplasm and a papillary stromal fragment partially covered in epithelium in a background of scant thin colloid. (Pap ×4)

Plate 4-7B, Pattern 7: Papillary carcinoma: crowded sheets of cells with denser cytoplasm, as well as branching papillary tissue fragments and swirling spiral tissue fragments with occasional psammoma bodies, thick colloid, and multinucleated histiocytes.

Plate 4-7C, Pattern 7: Papillary carcinoma showing highly cellular smear with large tissue fragments of epithelium with denser cytoplasm and crowded and variably overlapped pleomorphic nuclei and scattered nuclear pseudoinclusions. (Giemsa ×10)

Plate 4-7 Table
Pattern 7: Epithelial Tissue Fragments With Well-Defined Cytoplasm, Nuclear Grooves and Pseudoinclusions, and Variable Papillary Tissue Fragments and Variable Colloid.
Entities in the Differential Diagnosis

  • i.

    Papillary carcinoma

  • ii.

    Hyalinizing trabecular tumor

Plate 4-8
Pattern 8 Predominantly Single Cells and Epithelioid or Spindle Cells

Plate 4-8A, Pattern 8: Anaplastic carcinoma: This highly cellular smear shows predominantly dispersed cells with small irregular tissue fragments. (Pap ×2)

Plate 4-8B, Pattern 8: Various cell types including anaplastic carcinoma or metastatic carcinomas with glandular (in top right) or signet ring cell (in bottom right) differentiation, in a background of necrosis and large multinucleated tumor cells and pleomorphic large nuclei.

Plate 4-8C, Pattern 8: Anaplastic carcinoma: Highly cellular smear with irregular tissue fragments of crowded cells with some dispersed cells in the background. (Pap ×4)

Plate 4-8 Table
Pattern 8: Predominantly Single Cells With Epithelioid or Spindle Cells
Entities in the Differential Diagnosis

  • i.

    Medullary thyroid carcinoma

  • ii.

    Poorly differentiated ("insular") carcinoma of thyroid

  • iii.

    Anaplastic carcinoma of thyroid

  • iv.

    Metastatic tumors including melanoma and lobular breast carcinoma.

Thyroid Fine Needle Biopsy: Normal Components

Smear Pattern: Follicular Tissue Fragments and Fragments in Which Fibrovascular Strands Separate Follicles of Varying Size, in a Background of Variable but Usually Considerable Thin Colloid

Cytopathology

Cytologic diagnostic criteria ( Plate 4; Figs. 4-1 to 4-5 )

  • Pattern: Follicular tissue fragments and fragments in which fibrovascular strands separate follicles of varying size, in a background of variable but usually considerable thin colloid .

    Figure 4-1, Sheet of follicular epithelium showing regular nuclear arrangement, mainly round nuclei, and pale cytoplasm, in a background of thin colloid showing fine bubbling. (Pap ×40)

    Figure 4-2, Sheet of follicular epithelium with round, evenly spaced nuclei, minimal anisonucleosis, and pale, ill-defined cytoplasm, in a background of thin colloid that has focally washed off the slide. (Giemsa ×20)

    Figure 4-3, The stripped follicular cell nuclei lack cytoplasm, resemble lymphocytes, and aggregate focally. (Giemsa ×40)

    Figure 4-4, The colloid stains purple-blue in the Romanowsky stain. The red cells pushed together in strings of cells separated by thin colloid or clear gaps of previous colloid. There are scant follicular cells.

    Figure 4-5, Eosinophilic, thick colloid fragments are present with several minute sheets of follicular epithelium in a background of thin colloid showing small air bubbles from a colloid nodule. (Pap ×10)

  • Cellularity is low.

  • Follicular sheets with well-ordered honeycomb of cells with poorly defined fragile pale cytoplasm, and round, smoothly outlined nuclei.

  • Bare follicular nuclei: the same size as lymphocytes but lacking cytoplasm.

  • Thin colloid: violet to pale purple in the Giemsa stain, pale green with fine bubbles in the Pap stain, with folds and cracks and a distinct edge.

  • Thick colloid: dense fragments of angulated purple (Giemsa) material.

Skeletal muscle from strap muscles and ciliated columnar cells and hyaline cartilage from the trachea can be present as contaminants, usually associated with an immediate loss of vacuum during aspiration and a quick cough from the patient as the needle enters the trachea. The tissue fragments of follicles separated by stroma are rarely seen in normal thyroid.

The thick colloid has to be differentiated from amyloid, which is lighter staining, dense, and waxy, and collagen, which is fibrillar and lighter in staining. In difficult cases, Congo-Red stain of the smear or cell block can be helpful.

Suppurative Thyroiditis

Smear Pattern: Inflammatory Cells Predominate: Suppurative

Clinical Features

On rare occasions, patients can present with a diffusely enlarged or tender hot nodule in the thyroid associated with a febrile, debilitating illness, usually in a young patient or an immunocompromised patient. Clinically, the DD includes de Quervain thyroiditis or an inflamed lymph node. Drainage of any “cystic” component may be therapeutic.

The cause is usually a Streptococcus spp. or Staphylococcus aureus or gram-negative bacteria.

Cytopathology

Cytologic diagnostic criteria

  • Pattern: inflammatory cells predominate: suppuration .

  • Cellularity of the suppuration is high.

  • Large numbers of neutrophils in various stages of degeneration are present with some histiocytes and scant follicular epithelium.

  • Thin colloid is scant obscured by the pus.

  • Bacteria such as cocci may be seen in the Giemsa.

Disseminated fungal infection such as Aspergillus spp. or Cryptococcus neoformans may present with a suppurative granulomatous thyroiditis, so the Grocott methenamine silver stain and Gram stain are required, and correlation with cultures is mandatory.

Differential Diagnosis

The pus is distinctive. The possibility of anaplastic carcinoma might be considered.

Ancillary Diagnostic Studies

Cultures are obligatory.

Mycobacterial Thyroiditis

Smear Pattern: Inflammatory Cells Predominate: Granulomatous

Clinical Features

In countries with endemic tuberculosis, Mycobacterium tuberculosis has been reported as presenting with a thyroid nodule, usually cold and pain free.

Cytopathology

Cytologic diagnostic criteria

  • Pattern: inflammatory cells predominate: granulomatous .

  • Cellularity is usually low.

  • Epithelioid granulomas are present, rarely show central necrosis, and may contain Langhans multinucleated giant cells with irregular or horseshoe-arranged relatively uniform nuclei.

  • Caseating necrosis background, which is granular.

  • Variable colloid depending on the degree of necrosis.

  • Lymphoid infiltrate in which small lymphocytes predominate.

  • Variable but can be scant follicular epithelium showing varying degrees of degeneration.

Differential Diagnosis

The DD includes fungal infections, as well as de Quervain thyroiditis and Hashimoto thyroiditis. In tuberculous thyroiditis there may be necrosis and there are frequent epithelioid granulomas, but Ziehl Neelsen stain for acid-fast bacilli and Grocott methenamine silver stain for fungi are required on direct smears or cell block material.

Sarcoidosis can involve the thyroid, but the epithelioid granulomas are not associated with necrosis, the process is usually diffuse rather than nodular, and there are distinctive chest x-ray and serologic features to assist in the correct diagnosis.

Ancillary Diagnostic Studies

Cultures are mandatory with polymerase chain reaction (PCR) testing when epithelioid granulomas are found to exclude tuberculosis, where cultures will only be positive in up to 70% cases, and fungal infections.

De Quervain Thyroiditis

Smear Pattern: Inflammatory Cells Predominate: Granulomatous

Clinical Features

Also called subacute or granulomatous thyroiditis, de Quervain thyroiditis usually presents with a distinctive history of a flulike illness followed by a swollen painful thyroid, but there may be no definite history of fever and the swelling may be focal and the firm nodule can mimic carcinoma. Thyroid functions tests can show mildly increased T4 and T3, as well as increased thyroid-stimulating hormone (TSH).

Sonography usually shows a diffusely enlarged predominantly hypoechoic thyroid gland. Subacute granulomatous thyroiditis shows a low uptake of iodine on radionucleotide scintigraphy (radioiodine uptake), probably because of follicular cell destruction. CT scan shows the thyroid gland to have diffusely decreased attenuation.

Cytopathology

Cytologic diagnostic criteria ( Plates 4-1A-C ; Figs. 4-6 to 4-9 )

  • Pattern: inflammatory cells predominate: granulomatous .

    Figure 4-6, Subacute granulomatous thyroiditis: cellular smear shows plentiful inflammatory cells and numerous, large, multinucleated giant cells in a “dirty” background (Pattern 1). (Pap ×4)

    Figure 4-7, Subacute granulomatous thyroiditis: fragments of thick colloid and plentiful, large, multinucleated giant cells in a background of degenerate cells and lymphocytes (Pattern 1). (Giemsa ×20)

    Figure 4-8, Subacute granulomatous thyroiditis: tissue fragment of irregular, partially degenerate follicular cells, large multinucleated giant cells, and some thick colloid in a background of degenerate cells, occasional epithelioid histiocytes, and lymphocytes. (Giemsa ×20)

    Figure 4-9, Subacute granulomatous thyroiditis: mix of inflammatory cells, a large multinucleated giant cell, and a fragment of colloid. (Pap ×40)

  • Cellularity usually high.

  • Plentiful, often large multinucleated giant cells.

  • Follicular sheets show varying degrees of degeneration with irregular, often distorted degenerate hyperchromatic nuclei and apoptotic debris or “blue granules.”

  • Inflammatory background with debris, degenerate stripped nuclei, apoptotic fragments, thick and some thin colloid, lymphocytes, epithelioid histiocytes, and macrophages, often containing phagocytosed colloid.

Differential Diagnosis

Multinucleated giant cells and lymphocytes are seen commonly in papillary carcinoma, chronic lymphocytic thyroiditis (although the giant cells are usually smaller in size and fewer in number), mycobacterial thyroiditis (where they are seen along with distinct epithelioid granulomas and often caseous necrosis), and “palpation thyroiditis.” The follicular epithelium is degenerate with typically poorly defined lightly stained cytoplasm, in contrast to the oncocytic and more prominent and well-defined cytoplasm of “Askanazy” cells of chronic lymphocytic or Hashimoto thyroiditis.

The multinucleated giant cells must be distinguished from intact follicles, in which there is an even distribution of nuclei and a basement membrane may be discernible at the hard-edged margin of the follicle.

Chronic Lymphocytic or Hashimoto Thyroiditis

Smear Pattern: Inflammatory Cells Predominate: Lymphoid

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