Johan Fagan, Kathy Taylor & Ellen Bolding

Almost any mass or tumour requires cytological or histological diagnosis before a management plan can be formulated. Performing tissue biopsy of masses and lymph nodes of the head and neck fills many junior doctors with fear due to the complex anatomy and the vascular structures and nerves that traverse the head and neck. Yet diagnostic material can be safely obtained from most masses in the head and neck in an ambulatory care setting.

Techniques and pitfalls to obtain diagnostic material are presented in this chapter. Techniques include brush cytology, fine needle aspiration biopsy/cytology (FNAB/ FNAC), core biopsy, punch biopsy, and open surgical biopsy.

Tumours of the upper aerodigestive tract are ideally biopsied directly either trans-orally or transnasally. Subcutaneous tumours are first sampled by the least invasive technique; only if the diagnosis remains in doubt does one employ progressively more invasive biopsy techniques until a diagnosis is made. A typical diagnostic sequence is: FNAC - core biopsy - open surgical biopsy.

What do pathologists require to make a diagnosis?

Clinical detail: A pathologist’s differential diagnosis and types of pathological tests are informed by the clinical information; this is particularly important with FNAC.


Tissue biopsy

Pitfalls and caveats

Figure 1
Figure 1: Actinomyces surrounded by neutrophils
Figure 2
Figure 2: Cystic cervical nodal metastasis originating from squamous cell carcinoma of the tonsil

Fine needle aspiration cytology/biopsy (FNAC/FNAB)

This is a technique whereby a cytologic diagnosis is made on an aspirate of cellular material that has been collected through a small-calibre needle and smeared and fixed on a glass microscope slide. In the absence of an obvious primary tumour, FNAC is generally the 1st line investigation of a neck mass. Because the relationship of tumour cells to the basement membrane cannot be determined on cytology, the cytologist is unable to distinguish between high grade dysplasia (carcinoma in situ) and invasive carcinoma; this distinction can only be made on histological examination of a tissue biopsy that includes the basement membrane.

FNAC is particularly useful in the head and neck for the following reasons:

Equipment for FNAC (Figure 3)

Local anaesthesia

The authors do not use local anaesthesia as the additional needle stick for the anaesthetic also causes discomfort and the anaesthetic solution may obscure a small mass.

Figure 3
Figure 3: FNAC equipment: syringe, gauze swab, 2 glass microscope slides, 23-gauge needle, alcohol swab, fixative

Accessing the mass

FNAC is done either with the patient sitting in a chair, or lying down. With FNAC of the thyroid the neck may be hyper-extended over a bolster placed under the shoulders. Access to lymph nodes along the jugular chain may be improved by turning the head. If possible the mass is fixed between the fingers of the non-dominant hand.

Masses in Level 1b of the neck may be more accessible and better stabilised by displacing them inferolaterally with a finger placed in the lateral floor of mouth. Great care should however be taken not to pierce the operator’s finger in the mouth with the tip of the needle.

Ultrasound-guided (USGFNAC) or CT-guided FNAC may be done for masses that are difficult to access with confidence e.g. deep-seated masses in the deep lobe of parotid or parapharyngeal space, or smaller masses.

Harvesting the cellular material

Non-aspiration technique

Also known as the fine-needle capillary method, this is the authors’ preferred method. It relies on capillary action to draw sheared cells up a small-calibre needle. The non-aspiration technique is easier to perform than aspiration technique, improves the operator’s ability to direct the needle tip into a smaller mass, and is less likely to cause a bloody aspirate which is particularly advantageous with vascular structures such as thyroid gland.

Aspiration technique

The aspiration technique employs negative pressure generated by a syringe as well as the shearing effect of the needle, to aspirate cellular material from a mass. A pistol grip syringe holder can be used as it allows more uniform suction and makes it easier to direct the needle (Figure 4).

Figure 4
Figure 4b
Figure 4a, b: Example of pistol grip syringe holder

Smearing the aspirate

As soon as the aspirate has been ejected onto the glass microscope slide, a 2nd microscope slide is used to smear the cellular material into a monolayer of cells for microscopic examination (Figure 5).

Figure 6 illustrates two techniques that may be used to create a thin film. The “pull-push” smear technique is only applicable to liquid aspirates. The authors favour the so-called “crush” technique for FNAC of solid tumours which involves gently smearing the tissue between two opposing glass slides. 

Figure 5
Figure 5: Example of a smear showing oncocytic cells and lymphocytes that is too thick on the left, and a desired thickness (monolayer) on the right
figure 6
Figure 6: “Pull-push” (above) & “crush” (below) smear techniques

Fixing the smear

Smears are immediately fixed to avoid shrinkage artefact. This is achieved by either wet fixation or air-drying. Consult your cytopathologists about their preferred fixation method. As different stains are used for each technique, some prefer using both techniques for the same specimen, as the two methods can produce complementary results.

Wet-fixation technique

This is achieved by dipping the slide into 95% ethanol solution, or by applying a spray fixative (Figure 3). Spray fixatives typically consist of an admixture of polyethylene glycol and ethyl alcohol or isopropyl alcohol. The alcohol evaporates and leaves the glycol covering the smear. Material fixed by wet-fixation technique can be stained both with Papanicolaou (PAP) and haematoxylineosin (H&E) stains (Figure 7).

Wet-fixation technique

This is achieved by dipping the slide into 95% ethanol solution, or by applying a spray fixative (Figure 3). Spray fixatives typically consist of an admixture of polyethylene glycol and ethyl alcohol or       isopropyl alcohol. The alcohol evaporates and leaves the glycol covering the smear. Material fixed by wet-fixation technique can be stained both with Papanicolaou (PAP) and haematoxylineosin (H&E) stains (Figure 7).

Figure 7
Figure 7b
Figure 7: Examples of an H&E smears showing oncocytic cells and lymphocytes

Air-drying fixation technique

The smeared material is allowed to air-dry; drying should be quick, and may be aided with a hairdryer or a fan. The Wright-Giemsa stain is used for air-dried smears.


Label the slides with the patient's details and the origin of the aspirate. The slides in Figure 8 have been sandblasted at one end so that details can be written on the slide with pencil.

Figure 8
Figure 8: Sandblasted end of slides for labelling with pencil

Liquid based cytology

This technique is useful for hypocellular aspirates. Instead of being spread onto a glass slide, the material obtained by aspiration or by cytobrushing is transferred to a vial of fixative. The vial is then centrifuged and the sediment is used for the smear.

Exfoliative or brush cytology

Brush cytology is cheap, non-invasive, and virtually painless and requires minimal training. Although it is highly specific, it is less sensitive i.e. negative brush cytology does not rule out malignancy. It is therefore useful for screening suspicious oral lesions; if dysplastic cells or molecular alterations are identified, patients should be referred for tissue biopsy.

Figure 9 shows a close-up view of the tip of a cytobrush. A toothbrush is a reliable and cheap alternative (Figure 10).

Figure 9
Figure 9: Tip of cytobrush
Figure 10
Figure 10: Toothbrush is a reliable and cheap alternative for a cytobrush

Method of brush cytology

Trucut biopsy

Percutaneous core biopsy allows one to harvest a solid core of tumour for histological examination in an ambulatory setting. It is generally done with a Trucut biopsy system. Unlike FNAC, Trucut biopsy can seed tumour cells; therefore avoid using it for salivary gland tumours, melanomas etc. unless the tumour is considered to be inoperable.

An outer cannula unsheathes a sharp-pointed needle that can be retracted and advanced into and out of the cannula. The needle has a notch which traps a core of tissue which has been sliced off the tumour by the sharp end of the outer cannula (Figures 11a-d). It requires a two-handed technique, although automated and semi-automated systems are also available.