Keywords

Thyroid nodules are frequent among general population, although the rate of malignancy is frequently low, ranging from 3 to 7 % [1]. For such reason it is necessary to select the appropriate nodules for which surgery is required. Fine needle aspiration biopsy (FNAB ) is a diagnostic test routinely used in the evaluation of thyroid nodule disease and it is crucial to stratify the risk of malignancy. Furthermore, FNAB can also be used to evacuate large cystic nodules and is helpful in patients with thyroid cancer to detect lymph nodes metastases [2].

Many studies have proven the efficacy of FNAB in the management of patients with thyroid nodules with a reported sensitivity of 80–98 % and specificity of 58–100 % [3].

In the past, FNAB could be performed manually only on palpable thyroid nodules, while nowadays, because of the large diffusion of ultrasonography (US), it is possible to detect also nonpalpable nodules and such technique is helpful to identify suspicious nodules. In such situations the FNAB is performed under US guidance, which is the most widely diffused technique.

The first step to examine thyroid nodules is US. US represents the first line test to assess the location of the nodule, the size, the US pattern and to detect any possible suspicious lymph node. On the basis of US findings, considering the clinical history of the patient, the biochemical tests of thyroid function and possible information coming from radionuclide scan (thyroid scintigraphy or PET), the clinician has to select which nodules are eligible for FNAB .

The clinical characteristics that suggest performing FNAB are: patients with family history of thyroid cancer, story of neck irradiation, presence of a single, growing, fix and hard nodule, especially in young patients and in the male gender.

US suspicious features of thyroid nodules are: solid, hypoechoic, a “taller than wider” shape, irregular margins, absence of halo sign, microcalcifications, intranodular vascular flow.

Moreover, in case of a “cold” nodule, which has a decreased uptake of radionuclide in a conventional thyroid scintigraphy with 99-technetium or 123-Iodine, or in case of a focal uptake of FDG to PET scan, FNAB is suggested in order to exclude malignancy [4].

In presence of a multinodular goiter it is important to identify the most suspicious nodule, which is not always the dominant one. In fact, patients with multiple nodules have the same risk of malignancy as those with a single nodule. Thus, the sonographic features of each nodule should be assessed independently to determine the need for FNAB . If there are multiple coalescent nodules and none have suspicious US features, FNAB of the largest nodule is reasonable. In presence of complex nodules US guidance is essential to assess the biopsy on the solid part of the lesion, to reach a diagnostic conclusion as well as in inhomogeneous nodules US guidance is essential to biopsy the most suspicious part of the nodule.

There is no consensus among the scientific societies about the size of the nodule that can undergo FNAB .

The American thyroid association (ATA) guidelines published in 2009 [5] suggest performing FNAB in nodules: larger than 5 mm in patients with high risk history, larger than 1 cm in case of suspicious US features and larger than 2 cm in absence of suspicious US features. FNAB is not indicated in case of cystic nodule.

AACE/AME/ETA guidelines [2] published in 2010 suggest performing FNAB in nodules of any size in case of high risk patients or in case of US suspicious pattern.

The latest ATA guidelines (in press, presented at ATA satellite symposium at ICE/ENDO – June 2014) suggest performing FNAB in nodules: larger than 1 cm having high-intermediate US suspicious pattern; larger than 1.5 cm having US low suspicious pattern and larger than 2 cm having US very low suspicious pattern. In case of nodules smaller than 1 cm or cystic nodules, FNAB should never be performed.

This new approach is due to the fact that in the last three decades, overdiagnosis of clinically silent thyroid nodules by the widespread use of imaging methods has brought to an increase up to threefolds in the incidence of thyroid cancer, even though the rate of mortality has not changed. Thus far we have detected and treated thyroid microcarcinomas with questionable aggressiveness [6]. For this reason, routine FNAB is not considered cost-effective and an appropriate evaluation by an expert clinician is recommended before undergoing FNAB.

1 Procedure

FNAB is an invasive procedure and it is fundamental to obtain an adequate signed consent after the patient has been fully informed [7].

The patient must be advised about the procedure, aware of potential complications (usually self-limiting intra or extranodular bleeding), must be instructed not to swallow or speak during the insertion of the needle, must be informed of the need of taking serial samples on the same nodule, the time of the cytological result and the possibility of an inadequate sample, that may require the repetition of the sampling after at least 1 month (if possible to delay the repetition, on the basis of clinical history and US features). Correct information is also adjuvant to let the patient be relaxed during the procedure.

FNAB is a safe and minimally invasive procedure with low risks and can be usually performed on an outpatient basis by experienced physicians. In the past the procedure was performed manually, only on palpable masses, while nowadays US guidance is routinely used. In fact real-time US consents the visualization of the needle within the lesion enabling to reach targets as small as 2 mm, thereby facilitating accurate biopsy of small nonpalpable nodules. Furthermore, a color Doppler evaluation just before the procedure may help the operator to identify any large vessel in the surrounding tissue and choose the best way to sample the nodule, avoiding any vascular injury.

US is performed with a conventional high frequency linear array (7.5–14 MHz). Some arrays may also include specific supports used as needle guide. Such items may be useful in case of prolonged procedures (cyst evacuation, ethanol injection) more than in routine sampling.

A review of recently published data regarding thyroid cancer detection at US-guided FNA indicates a sensitivity of 76–98 %, a specificity of 71–100 %, a false-negative rate of 0–5 %, a false-positive rate of 0–5.7 %, and an overall accuracy of 69–97 % with the use of this method [8]. The false-negative rate with palpation-guided FNA (1–3 %) was higher than that with US-guided FNA (0.6 %) [9]. The literature reveals great variability in specimen cellularity [9]. Although the rate of specimen inadequacy with US-guided FNA is lower than that with palpation-guided FNA, US-guided FNAB yields an inadequate specimen in 10–20 % of procedures, perhaps because of the absence of uniformly adopted or standardized criteria for adequacy of thyroid FNAB specimens and specimen procurement techniques [10].

It is useful to have a proper tray including materials for topical cleaning, transducer covers, coupling gel and a wide set of needles. The needles normally used may be small (25–27 gauge) or medium (21–23 gauge).

The patient is placed in a supine position with the neck slightly extended, with a pillow or a pad placed beneath the patient’s shoulders. Anesthesia is not routinely performed. Some operators use topical lidocaine gel or patch, ethyl cloryde spray (that may anyway irritate the skin). In selected cases, especially for long procedures or for the use of large needles, 1–2 ml of 1 % lidocaine solution can be injected subcutaneously. The US monitor must be placed optimally for the operator’s view and the US array should be covered with a sterile mean, applying a coupling gel between the array and the sterile mean. The skin in the projective area of the thyroid nodule must be cleaned with an alcoholic solution.

The needles normally used in FNAB rise from 22 to 27 gauge. There is a correlation between the gauge of the needle and the cellularity of specimen, in fact the thinner the needle used, the higher the rate of sufficiency of cytological material. Special needle which are longer may be used for posterior nodules. Each needle may be attached to a 2–20 mL syringe, with an eventual syringe holder, according to the preference of the operator.

A freehand biopsy technique is the widest used technique. The syringe can be attached to the needle and placed just above the transducer. The needle may be introduced parallel or perpendicular to the transducer, and the needle tip should be carefully monitored during the procedure. All needle movements should be continuously visualized in real time. Poor needle visualization is a common difficulty. If the needle is parallel to the transducer, it will be visible in its entirety. However, if the needle is inserted at a steep angle, as it must be to reach deep lesions, or if it is inserted perpendicular to the probe, localization of its tip is more difficult (Fig. 2.1). The tip of the needle is visible only as a bright echogenic focus on the monitor as the tip bisects the scanning plane. If the needle tip is not visible, the position of the needle and transducer should be adjusted until the tip points toward the center of the lesion. When the needle reaches the target, the biopsy is performed.

Fig. 2.1
figure 1

An US-guided FNAB with perpendicular approach. Only the tip of the needle (AGO-arrow) is visible within the nodule as a bright echogenic focus on the monitor

Full size image

Biopsy specimens may be obtained with two widely used acquisition methods. The choice between “nonaspiration” and “aspiration” is a matter of operator preference.

The “needle only” technique or “nonaspiration” technique is based on the principles of capillarity action of cellular material into the needle without aspiration. The needle is withheld with two fingers and inserted in the nodule. After that, the operator should kindly move the needle up and down for a couple of times and may perform rotations of the needle. “Nonaspiration” FNAB is less traumatic and even more rarely associated with complications. The amount of the sample is directly proportional to the time of stay of the needle inside the lesion. After the procedure, the needle is attached to a syringe to extrude the material on the smear or in the vial. Such technique should be modified in case of mixed, hemorrhagic or cystic lesions.

The “aspiration” technique or “closed suction” is characterized by the freehand introduction under US guidance of a needle with a syringe placed on it. When the needle is inside the lesion, the syringe plunger is withdrawn to create a negative pressure and induce aspiration. The needle is moved up and down, the plunger is withdrawn, inside the lesion as long as the needle is taken out of the skin. At that moment the plunger can be released. This technique is very useful to perform the aspiration of fluid lesions.

At present there is no preference between the two techniques, and the choice is based on the feeling and the experience of the operator.

It is recommended that aspiration is performed at least twice at US-guided FNAB because of the fine caliber of the needle, but the number of aspiration can range from one to five aspirations. Multiple punctures of the nodule can ensure samples from different parts of the lesion. In case of complex nodules, with large fluid parts, it is necessary to increase the number of aspirations to obtain adequate samples.

After the procedure, a firm pressure is applied to biopsy site with gauze pad. Once bleeding has stopped, a sticking plaster is placed on the puncture site, the patient is observed for a few minutes and, if there are no problems, allowed to leave. The patient should be instructed to contact hospital staff or seek the emergency room if neck swelling occurs on the way home or at home.

The material collected during FNAB can be processed in two different ways.

Conventional smear (CS) has been the standard diagnostic method for detecting thyroid lesions for many years, as it is cheap, widely codified, quick and easy to be performed. Specimen is suddenly placed on a conventional smear by depositing the needle contents onto a glass slide, then smearing the material. Each slide is then fixed with alcohol prior to staining. When the Papanicolaou staining method is used, the smears should be quickly placed in 95 % ethyl alcohol. When Diff-Quik or Giemsa stain is used, the smear should simply be allowed to air dry. Papanicolaou staining is most commonly used for cytologic analysis of thyroid specimens, and it provides the clearest depiction of nuclear chromatin, ground-glass nuclei, and nuclear groove characteristics in papillary carcinoma. Diff-Quik or Giemsa stain helps visualize the characteristics of cytoplasm and colloid.

Another method for preparing slides is to rinse the contents of the needle into a cell suspension (in the procedure room), which is then used to make liquid-based thin-layer slides, or cell block material (in the laboratory). Liquid-based thin-layer slides are stained with Papanicolaou stain and processed in a cytology laboratory. Cell block material is stained with hematoxylin-eosin stain and processed in a histology laboratory [11].

Some authors have suggested that thin prep is diagnostically superior to conventional preparation in certain nongynecologic specimens, but this technique is quite expensive, as it is necessary to have dedicated laboratory tools and pathologist who are familiar with these cytological samples.

Thin prep has many advantages compared with conventional smears, including ease of preparation by the clinician, more consistent specimen quality, uniform specimen collection, convenient transportation from remote sites, decreased needle handling, preservation of nuclear detail, decreased screening time, and the ability to perform ancillary testing. Many authors have shown that thin prep has diagnostic equivalence to conventional smears but a well-trained pathologist with the newest method is essential because cytologic findings are quite different in the two preparations with regard to amount and character of colloid, architectural disruption, background elements, and nuclear details [12].

The final cytopathologic finding is generally reported using the Bethesda criteria [13], in which a sample is considered adequate if it contains a minimum of six groups of well observed follicular cells, with at least 10 cells per group.

The increasing diffusion of liquid-based thin layer slides has increased the possibility of performing further analysis on the aspired samples to increase sensitivity and specificity of the test, especially in case of undetermined lesion. Such analysis varies from immunocytochemistry to genetic markers. In the recent years there has been a wide diffusion of new marker in thyroid cytology. As to immunocytochemistry, the most diffused markers are galectin-3, HBME-1, CK-19 and CD56, which can be used in combination.

As to molecular markers , the most used genes analyzed are BRAF, RET/PTC and RAS. In the most recent years there are at least two commercial multigene classifiers that can be applied to the FNAB samples to produce risk stratification on the basis of gene profile. Such tests still need to be proved before becoming part of a routine evaluation in the clinical practice [14, 15].

In case of inadequate samples, it can be considered to repeat the procedure no sooner than 1 month (if the US pattern of the nodule is not very suspicious), in order to prevent false-positive interpretation due to biopsy-induced reactive/reparative changes, and if available, it can be useful to have an on-site cytologic evaluation.

In case of repeated inadequate samples, other techniques have been suggested to define the diagnosis of the nodule. The most suggested technique is core-needle biopsy. The 21 gauge needle is inserted into the nodule under US guide in freehand fashion. When the tip is inside of the nodule, mandrel is removed and the needle is advanced within the nodule to obtain a tissue core. Needle is moved ahead across the nodule’s margin reaching extranodular tissue. Then, the needle is removed. The obtained core sample is fixed by buffered formalin 10 % [16].

Although FNA is an established test for the evaluation of thyroid nodules with high sensitivity and accessibility, as we mentioned earlier, in some cases it does not yield sufficient diagnostic material even with repeated attempts and scant aspirates or those with borderline adequacy may be a source of diagnostic error. Core-needle biopsy provides a large histologic core of tissue, which may have a greater effect on surgical decision making than the cytologic diagnosis. For these reasons, a core-needle biopsy is considered for patients in whom FNAB produces only specimens of grossly scant-appearing cellularity after several passes or in patients who return for a repeat biopsy after a nondiagnostic initial FNAB.

FNAB is used as therapeutic procedure to evacuate large cystic lesions. After evacuation, it is necessary to monitor the lesion with US, for eventual recurrence. For those patients with subsequent recurrent symptomatic cystic fluid accumulation, surgical removal or percutaneous ethanol injection (PEI) are both reasonable strategies, based on compressive symptoms and cosmetic concerns. The PEI procedure consists in percutaneous intralesional ethanol injection, which induces dehydration followed by coagulative necrosis and vascular thrombosis and occlusion. Volumes of 0.4–2 ml of ethanol are injected, and patients may receive repeated treatments. The technique requires a well-trained staff. Transient, sometimes severe, local pain is the most frequent side effect, followed by transient fever, and occasionally transient dysphonia.

2 Contraindications

The only serious contraindication is hemorrhagic diathesis . No routine clotting screening are required before FNAB , but in case the patient is on anticoagulant drugs, these must be interrupted at least 5 days before the procedure, shifting the treatment to heparin, under medical supervision. The procedure can be performed only when the clotting parameters are normal.

The use of antiplatelet drugs is not a full contraindication to FNAB , but it increases the risk of bleeding and inadequate samples. For that the clinician should evaluate the risk of a possible interruption of such treatment 3–7 days before the procedure. In such situation it could be useful to defer the procedure and contact the referring physician of the patient to balance the risk.

In case of specific hematological disorders (such as hemophilia or thrombocytopenia), it is necessary to have a specific specialist consult before the procedure.

Furthermore, it is necessary that the patient can lay recumbent in the appropriate position for the biopsy, with an adequate neck hyperextension. In case of old patients or patients with orthopaedics, neurological, or ear disorder such limitation can be so severe that the procedure may result impossible. The patient must also be cooperative with the operator to stand still without swallowing or breathing deeply.

3 Complications

The possible complications of the procedure can be; hemorrhage, caused by accidental microvascular trauma; large hemorrhage is rare, but the risk is always high, especially in case of nodules adjacent to the vessels (carotid and jugular); pain, due to the skin, muscle and thyroid capsule innervation. Also an accidental puncture of the trachea causes an acute stinging pain. The risk of hemorrhage and pain is increased in case the patient does not stand still during the procedure.

Conventional bleeding and small hemorrhage can be dominated by compression and/or an ice pack. The operator should look for eventual internal bleeding before the patient’s departure.

The risk of bacterial infection causing acute thyroiditis is very rare but it has been described in literature. For that it is important to monitor patients with higher risk of infection (AIDS, leucopenia) [17].

4 Thyroglobulin in Washout Fluid from Lymph Node FNAB

During the follow-up of patients affected by differentiated thyroid cancer (DTC), serum thyroglobulin assays and neck US are routinely recommended for the surveillance of recurrences [5]. Although the criteria for US to distinguish benign from metastatic lymph nodes have been described, its specificity is not optimal. The US criteria for possible malignant infiltration of lymph nodes included: a rounded rather than oval shape, with a long-to-short axis ratio inferior to 1.5, irregular internal echogenicity, calcifications, loss of the fatty hilus peripheral vascularity, and cystic change [5, 18].

To detect and evaluate cervical lymph nodes in patients with thyroid malignancy, US and FNAB have been standard diagnostic modalities. Although the accuracy of US diagnosis of node metastasis has improved, FNAB shows disappointing sensitivity, varying from 75 to 85 % with a high rate of nondiagnostic samples or even provide false-negative results, especially in small or cystic metastatic lymph nodes [19, 20]. To improve the diagnostic yield of FNAB, direct measurement of the concentration of thyroglobulin in the washout fluid of the needle is used in FNAB, since its first description in 1992 [19].

The measurement of thyroglobulin in the washout fluid of lymph node FNAB has been proposed to be a useful diagnostic method in detecting metastatic lymph nodes of DTC patients. Several studies have reported thyroglobulin in the washout fluid of FNAB to be more sensitive than FNAB for detecting metastasis, and the accuracy of FNAB is improved when combined with thyroglobulin in the washout fluid.

US guided FNAB is performed on suspicious lymph nodes by an experienced operator using a 21–25 gauge needle attached to a 10 mL syringe. The technique is the same as thyroid FNAB previously described. Each lesion should be aspirated at least twice. The samples can be placed on glass smears or using thin prep methods for cytological examination. The same needle and syringe must be rinsed with 0.5–1.0 ml of normal saline to a final volume of 1.0 ml and the washout submitted for measurement.

Thyroglobulin assays in the FNAB washout can be performed using electrochemiluminescence (ECLIA) immunoassay, that is conventionally standardized for serum thyroglobulin and results are expressed in “ng/mL”. Results are dependent on accurate sampling and a maximum needle wash volume of not more than 1.5 ml.

However, the cutoff value of thyroglobulin in the washout fluid remains controversial on account of differences in sample treatments and thyroglobulin assays. There is no established diagnostic cut off, particularly for the latest generation of highly sensitive thyroglobulin assays [21]. Several cut off have been considered in literature both on the absolute level and as ratio respect to serum thyroglobulin (especially in case of patients with high levels of thyroglobulin on LT4). The role of thyroglobulin in the washout fluid in patients with positive antithyroglobulin antibodies is controversial. Furthermore, the use of thyroglobulin in the washout fluid in the evaluation of suspicious cervical lymph nodes is appropriate in selected patients, but interpretation may be difficult in patients with an intact thyroid gland, as diagnostic test in the preoperative phase.

The same technique can be used also to detect calcitonin in the washout fluid of FNAB of thyroid nodule suspicious for medullary thyroid carcinoma or lymph nodes FNAB in patients affected by medullary thyroid cancer [22]. The limits of the test are the same as thyroglobulin assay. The evaluation of PTH in the washout fluid of FNAB is used to identify lesions suspicious for parathyroid glands or cysts.