- Email: [email protected]
Principles and techniques of biopsy with special reference to fine-needle aspiration cytology
Basic science
Principles and techniques of biopsy with special reference to fine-needle aspiration cytology
gastritis visualized by endoscopy). The main biopsy techniques in clinical practice in the UK are open biopsy, core biopsy, endoscopic biopsy and fine-needle aspiration (FNA). This contribution will focus on FNA because it has a central role in the surgical management of superficial lesions (e.g. breast, thyroid gland). It can also be used to remove diagnostic material from deep-seated lesions of organs (e.g. lung, liver) under image guidance (Figure 1). Recent advances in ultrasound, CT and MRI have led to a wide variety of internal structures (e.g. mediastinum, pancreas) becoming amenable to FNA.
John R Mitchard Kathleen E Romain Neil A Shepherd
History Reports of FNA first appeared in the medical literature in the first half of the nineteenth century following the work of Hodgkin (London, UK) amongst others. The procedure fell in and out of favour until the late 1960s, when a Scandinavian study (correlating the histological and cytological appearances of nearly 3500 consecutive palpable breast lesions) was published, showing FNA cytology to be a useful diagnostic tool. In the year to March 2002, 89% of breast cancers operated on in the UK had been diagnosed preoperatively and FNA cyto logy alone was responsible for a significant proportion of these. Thus, the use of intraoperative frozen section in the management of breast cancer in the UK has declined significantly and the patient is now involved in the planning of definitive surgery.
Abstract Fine-needle aspiration (FNA) cytology is an inexpensive, effective and (generally) safe procedure for diagnostic sampling of superficial masses. In the UK, it has attained central importance in the management of palpable lesions of the breast and thyroid gland. The use of image guidance means that aspiration of impalpable and deep-seated lesions is routine in radiological practice in the UK. The aim of the FNA procedure is to collect sufficient representative material from the lesion to enable a cytological diagnosis—i.e. based on the appearance of dispersed individual cells and cell groups—to be made. As well as its role in the primary diagnosis of tumours, FNA cytology in very useful in the staging of malignant tumours. For example, endoscopic ultrasound-guided FNA of a paraoesophageal lymph node can detect metastatic oesophageal carcinoma. Another useful role for FNA is in diagnosing recurrence of malignancy, particularly lymphoma. Clinical FNA procedures are described and potential causes of an inadequate—i.e. non-diagnostic—aspirate are discussed. Various modalities of image guidance are discussed and methods of slide preparation compared. The role of special techniques such as immunocytochemistry and fluorescence in situ hybridization is illustrated by means of diagnostic scenarios. The uses and limitations of FNA cytology of the breast, thyroid, lymph nodes and salivary glands are discussed.
Aim The aim of FNA is to collect sufficient representative material from the lesion to enable a cytological (i.e. based on the appearance of dispersed individual cells and cell groups) diagnosis to be made. This contrasts with a histological diagnosis (i.e. based on the appearance of cells and their arrangement within tissue).
FNA versus tissue core biopsy (Table 1)
Keywords breast; FNA cytology; lymph node; pathology; thyroid
The use of FNA can obviate the need for tissue core biopsy or more invasive tissue sampling. This is particularly important in Biopsy is the removal of tissue from a living subject in order to establish the presence or extent of a disease. Sometimes, tissue may be taken to detect the cause of a disease (e.g. causative Helicobacter pylori may be identified in a biopsy from an area of
John R Mitchard MRCPath MPH is a Consultant Histopathologist and Cytopathologist at the Royal Cornwall Hospital Trust, Truro, UK. Conflicts of interest: none declared. Kathleen E Romain APCP CYTO is a Consultant Cytopathologist at Cheltenham General and Gloucestershire Royal Hospitals, Gloucestershire, UK. Conflicts of interest: none declared. Neil A Shepherd DM FRCPath is a Consultant Histopathologist at Gloucestershire Royal and Cheltenham General Hospitals, Gloucestershire, UK, and Visiting Professor at Cranfield University, London, UK. Conflicts of interest: none declared.
SURGERY 26:2
Figure 1 A fine needle passes horizontally into a mass lesion in the liver under CT guidance.
38
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
pneumothorax is not an infrequent occurrence following FNA of the lung. The risk of seeding tumour along the needle path appears to be very small.
FNA versus tissue core biopsy Feature
FNA
Tissue core biopsy
Speed of reporting
Results possible on same day Not needed (generally) Usually minor
In general, result obtained the following day May be needed May be serious
Common
Less common
Low Large numbers of cells Not preserved Can be done at bedside Not usually
Higher Smaller numbers of cells Preserved Difficult at time of biopsy Usually
Anaesthesia Complications of procedure Inadequate specimens Cost Cellular sampling Tissue architecture Assessment of adequacy of sample Spare tissue for special tests
Technique Operator Radiologists will perform most image-guided aspirates, but physicians, surgeons and cytopathologists may participate in the FNA of palpable lesions. Practice varies between centres in the UK. Some cytopathologists perform all clinical aspirates of breast and thyroid lumps, whereas all aspirates are performed by surgeons in other centres. All operators must be suitably trained and their performance monitored. Superficial aspirates In most situations, a 21-gauge (green) needle is attached to a 20 ml syringe, leaving 5 ml of ‘dead space’ in the syringe (which permits expulsion of the material onto the slide and eliminates the need to detach and reattach the needle in order to introduce air). The skin is cleaned with an alcohol swab and the lesion held between the thumb and index finger of the non-dominant hand. The tip of the needle is advanced into the lesion and 6–10 ml of suction is applied to the syringe (a syringe holder may be beneficial). Several passes are made through the lesion in various planes, the needle being rotated between each pass. The suction is released and the needle withdrawn when a drop of blood or fluid is seen in the needle hub. It is the capillary action of the hollow needle (rather than the negative pressure applied by the syringe) that is responsible for sampled material moving into the bore. It is sometimes preferable to use a needle without a syringe, especially in highly vascular sites like the thyroid gland. The role of suction is to empty the needle and allow necrotic material or cyst fluid to be removed from masses. The needle should be removed and firm pressure applied to the area if excessive bleeding is noted. It may then be possible to re-attempt the aspiration from a different angle. Otherwise, FNA can be performed one week later. According to the preferences of the reporting cytopathologists, the sample can be spread onto a slide, air dried and/or fixed or injected into transport medium. The causes of non-diagnostic aspirates are given in Table 2.
Table 1
advanced malignancy when a tissue diagnosis is needed to plan palliative management. FNA is a relatively inexpensive procedure compared to tissue core biopsy because less laboratory time and fewer reagents are consumed in preparing the slides. Cost minimization is very important because FNA is used widely in the evaluation of breast lesions detected as part of the UK Breast Screening Programme. Many centres in the UK operate ‘one-stop’ breast clinics (in which women with breast lumps are assessed clinically, radiologically and cytologically) because stained slides of FNA specimens can be prepared in a short time, enabling rapid diagnosis (often on the same day). FNA is a convenient, relatively painless and inexpensive procedure, but it must not be used indiscriminately. The aspiration of impalpable or non-existent lesions in clinic will frequently yield smears that are non-diagnostic or misleading, leading to a perception that FNA is a poor diagnostic tool. Clinicians must be aware of the limitations of cytological dia gnosis. For example, FNA cannot distinguish between: • in situ and invasive carcinoma of the breast • a follicular adenoma and follicular carcinoma of the thyroid gland (see below).
Risk
Possible causes for a non-diagnostic FNA
In general, FNA of superficial lesions is a safe procedure; bleeding is the main complication. Puncture of adjacent structures (e.g. the trachea in thyroid aspiration) occurs occasionally, but is usually straightforward to manage. Other risks include puncture of the jugular vein, carotid artery and apical pleura in the supraclavicular fossa. These may be avoided by detailed knowledge of anatomy and landmarks, and careful palpation of the lesion on examination of the patient. Aspiration of deeper structures carries risks associated with the path of the needle. For example,
SURGERY 26:2
• Lesion does not exist • Needle misses lesion • Aspiration of contents of cystic/necrotic lesion • Haemorrhage obscures diagnostic material • Low yield of cells from a sclerotic neoplasm • Inexperienced operator Table 2
39
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
The slides are air dried or fixed immediately with alcohol. Air-dried smears can be stained with May–Grunwald–Giemsa or Diff-Quik™, allowing rapid staining and assessment of the smear within minutes of the aspirate being taken. Thus, a cytopathologist can evaluate the smear, if adequate cellular material has been sampled, before the patient leaves the department. This is particularly important for deep-seated lesions, where an inadequate aspirate would entail another session in an already overstretched radiology department. May–Grunwald–Giemsa or Diff-Quik™ stains are the best for demonstrating cytoplasmic detail and stain colloid particularly well in thyroid aspirates. Papanicolaou stain is used most commonly for fixed slides and shows nuclear detail very clearly.
Deep/image-guided FNA Aspiration of deep-seated lesions is predominantly the domain of the radiologist and a detailed discussion is outside the scope of this contribution. The chosen modality will usually be that which enables the FNA to be performed most quickly because most radiology departments in the UK have huge demands placed on CT and MRI scanners. Ultrasound allows real-time guidance of the needle with imaging in any plane. Use is limited because ultrasound is not transmitted through air or bone. The needle is manipulated with one hand while the other hand holds the transducer so that the tip of the needle can be visualized. Ultrasound-guided FNA is a technically demanding procedure. Endoscopic ultrasound-guided fine-needle aspiration biopsy allows guidance of an aspiration needle placed endoscopically into the pancreas, a mediastinal lymph node or perigastric lymph node via the wall of the duodenum, oesophagus or stomach. Diagnosis of pancreatic masses and staging of gastric and oesophageal cancer is the most common indication for this investigation.
Indirect smears are usually prepared in the cytology laboratory. Following aspiration, needle washings are prepared at the bedside. Transport medium or fixative is aspirated via the attached needle into the syringe and washed back into the transport vial for centrifuging in the pathology department. This method offers the possibility of spare slides being available for special stains (including immunocytochemistry). May–Grunwald–Giemsa stains cannot be performed unless a separate FNA pass is performed to prepare an air-dried smear. Direct communication with the pathologist is recommended if the laboratory does not have clear guidelines for the preparation of FNAs.
Fluoroscopy is used for real-time guiding of a needle into lesions that are visible in two planes. The needle is inserted along the axis of a central X-ray beam and an orthogonal beam assesses the proximity of the tip of the needle to the lesion. Lung lesions (especially those in the lower zones) are usually amenable to FNA guided by fluoroscopy.
Special techniques in the laboratory The use of special laboratory techniques in FNA is reliant on cytology having adequate excess cellular material in the form of needle washings or multiple fixed slides prepared directly.
CT scanning allows sampling of small lesions almost anywhere in the body; the lung, liver (Figure 1) and retroperitoneum are the common sites. A vertical or horizontal trajectory is used, allowing the full length of the needle to be visualized (Figure 1). The passage of a fine needle through abdominal structures (e.g. small intestine) does not appear to cause problems. However, the colon should be avoided because of the risk of spreading bacteria along the needle track.
Immunocytochemistry is used widely in histopathology because abundant excess material is usually available. Material is often limited in FNA samples, so antibodies must be chosen carefully. The cytological appearances (along with the radiological and clinical findings) should be considered to produce a differential diagnosis. For example, FNA of a lung mass may reveal an adenocarcinoma. In a middle-aged female, this might represent a primary tumour of the lung or a metastasis from the breast. Immuno staining with antibodies to thyroid transcription factor-1 and oestrogen receptors may distinguish between the two. Diagnosis of metastatic melanoma or carcinoma and subtyping of malignant lymphoma are other instances where immunocytochemistry might be useful.
Slide preparation Following aspiration, most of the cellular material is within the needle bore. The material is expressed onto the glass slide using the residual 5 ml of dead space in the syringe after suction is released. Direct smears are the conventional method for preparation of aspirated material. A slide is labelled with the patient’s details and the needle contents gently expressed onto one end of the slide. A single layer of cells can be produced on the slide without crushing them by spreading the material with the needle or by using a second glass slide. The second glass slide can be held at an angle of about 30–40° to the first slide and the end brought into contact with the droplet (which is allowed to distribute along its edge). The second slide is then drawn along the first in a manner similar to that used to produce a blood film. Alternatively, the material can be gently ‘sandwiched’ between two slides that are then drawn along each other’s lengths to produce two smears; this method is suitable for ‘dry’ aspirates.
SURGERY 26:2
Fluorescence in situ hybridization can be applied to relatively small numbers of interphase cells, so it can be applied to FNA cytology. Fluorescence in situ hybridization can help to diagnose subtypes of non-Hodgkin’s lymphoma and sarcomas by detecting specific chromosomal translocations. HER-2/neu alterations in breast cancer aspirates can also be detected by this method.
Specific sites Breast Although the use of tissue core biopsy is increasing, FNA cyto logy is central in the diagnosis of breast cancer. 40
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
Thyroid In general, FNA is a first-line tissue investigation for solitary thyroid nodules and is also useful in the context of clinically and radiologically malignant tumours, when potentially treatable lymphomas can be distinguished from anaplastic carcinomas. All personnel involved in the taking and reporting of thyroid FNAs (and those acting upon the reports) must be aware of the limitations of the procedure. The patient is positioned supine with a pillow behind the shoulders (allows the neck to extend and the sternocleidomastoid muscles to separate). A 23-gauge (blue) needle should be used because the gland is highly vascular. For most lesions, three passes in a single plane usually offers the best compromise between sampling sufficient cellular material and causing excess contamination by blood. Some sclerotic tumours may need more passes. Local complications include haemorrhage, carotid puncture and tracheal puncture: the latter may cause coughing and haemoptysis (rare). Sampling without suction may be helpful for lesions where previous FNA has caused excessive blood contamination. The majority of solitary nodules will be dominant colloid nodules in a multinodular goitre or follicular adenomas. A minority will be follicular carcinomas, papillary carcinomas and other tumours (including anaplastic and medullary carcinomas). It is not possible to distinguish between follicular adenoma and carcinoma on cytological grounds because the distinction is based
FNA cytology scale of the breast C1: Inadequate (<5 groups of epithelial cells) C2: Benign C3: Atypia (probably benign) C4: Suspicious of malignancy C5: Malignant Table 3
Stereotactic FNA is commonly used for the sampling of small lesions. Mammograms are taken from two angles in order to calculate the coordinates of the lump. The position of the needle holder is set accordingly. FNA cytology of the breast is reported on a scale of C1 to C5 (Table 3). Benign lesions of the breast have cohesive groups of epithelial cells in a background of bare myoepithelial cell nuclei (Figure 2a), whereas the cells of malignant aspirates are dys cohesive and exhibit nuclear pleomorphism (Figure 2b and c). Clinical examination and radiological findings are similarly scored. A tissue core biopsy is performed if there is discrepancy between the three arms of the assessment. A tissue core biopsy has the advantage of: • providing sufficient material for hormone receptor studies • allowing distinction between in situ and invasive carcinoma.
FNA cytology of the breast a
b
c
a The smear shows the characteristic cytological features of a benign lesion. There is a large group of benign epithelial cells showing a ‘staghorn’ morphology; numerous bare/stripped nuclei (myoepithelial cells) are in the background. The lesion was a fibroadenoma and the cytology is distinctive for this diagnosis. b The smear is taken at the same magnification as a and shows a malignant lesion characterized by dyscohesive epithelial cells. c The smear is the same sample as b, but taken at a higher magnification to show the irregular pleomorphic nuclei in the epithelial cells. Figure 2
SURGERY 26:2
41
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
Correlation of FNA and histology in papillary carcinoma of the thyroid gland a
b
a FNA cytology shows enlarged thyroid epithelial cells with an obvious nuclear inclusion. b Subsequent histology shows a tumour composed of papillae (finger-like processes lined by epithelium with a fibrovascular core) lined by epithelial cells with pleomorphic, open nuclei.
Figure 3
on the histological presence (carcinoma) or absence (adenoma) of capsular and vascular invasion. The initial treatment for both neoplasms is lobectomy and so the FNA is generally reported as being consistent with a follicular lesion. It is often impossible to distinguish cytologically between a follicular neoplasm and a colloid nodule. Papillary carcinoma has classic nuclear features (Figure 3a and b) and treatment is total thyroidectomy. Attempts to formalize the reporting of FNAs of the thyroid into five categories (in a similar system to that used for the breast) have not met with universal acceptance.
nodes are not unusual in squamous cell carcinoma of the tonsil or posterior tongue; however, these aspirates may be misinterpreted as branchial cysts. The diagnosis of lymphoma by FNA allows planning of formal lymph node biopsy and staging procedures. Formal biopsy allows assessment of lymph node architecture and provides abundant material for immunocytochemical assessment. FNA can provide material for ancillary tests (e.g. flow cytometry, immunohistochemistry), but full categorization of the tumour at the time of primary diagnosis is desirable because it provides archive material for further tests, should new treatments become available. FNA is extremely useful for diagnosing recurrences.
Lymph node FNA of a lymph node is useful in the management of lymphadenopathy, enabling the diagnosis of reactive lymphadeno pathy and suggesting specific conditions (e.g. tuberculosis, toxoplasmosis); supplementary investigations (e.g. Mantoux testing, toxoplasma serology) can then be suggested. Excision biopsy can be undertaken if no cause is found and lymphadenopathy persists. The diagnosis of metastatic malignancy is important to recognize because disseminated disease is often actively managed. The appearance of the tumour cells may suggest a primary site. Necrotic metastases in cervical lymph
SURGERY 26:2
Salivary glands The major salivary glands are readily accessible with a needle and FNA of masses is usually attempted. Pleomorphic adenoma is the most common tumour of the major glands. Determining whether a mass is non-neoplastic (e.g. a focus of chronic sialadenitis), benign or malignant can influence the nature of the definitive surgery (or whether surgery takes place). For example, Warthin’s tumour (adenolymphoma) is a benign tumour most often seen in the elderly and surgery may not be necessary. ◆
42
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Principles and techniques of biopsy with special reference to fine-needle aspiration cytology
gastritis visualized by endoscopy). The main biopsy techniques in clinical practice in the UK are open biopsy, core biopsy, endoscopic biopsy and fine-needle aspiration (FNA). This contribution will focus on FNA because it has a central role in the surgical management of superficial lesions (e.g. breast, thyroid gland). It can also be used to remove diagnostic material from deep-seated lesions of organs (e.g. lung, liver) under image guidance (Figure 1). Recent advances in ultrasound, CT and MRI have led to a wide variety of internal structures (e.g. mediastinum, pancreas) becoming amenable to FNA.
John R Mitchard Kathleen E Romain Neil A Shepherd
History Reports of FNA first appeared in the medical literature in the first half of the nineteenth century following the work of Hodgkin (London, UK) amongst others. The procedure fell in and out of favour until the late 1960s, when a Scandinavian study (correlating the histological and cytological appearances of nearly 3500 consecutive palpable breast lesions) was published, showing FNA cytology to be a useful diagnostic tool. In the year to March 2002, 89% of breast cancers operated on in the UK had been diagnosed preoperatively and FNA cyto logy alone was responsible for a significant proportion of these. Thus, the use of intraoperative frozen section in the management of breast cancer in the UK has declined significantly and the patient is now involved in the planning of definitive surgery.
Abstract Fine-needle aspiration (FNA) cytology is an inexpensive, effective and (generally) safe procedure for diagnostic sampling of superficial masses. In the UK, it has attained central importance in the management of palpable lesions of the breast and thyroid gland. The use of image guidance means that aspiration of impalpable and deep-seated lesions is routine in radiological practice in the UK. The aim of the FNA procedure is to collect sufficient representative material from the lesion to enable a cytological diagnosis—i.e. based on the appearance of dispersed individual cells and cell groups—to be made. As well as its role in the primary diagnosis of tumours, FNA cytology in very useful in the staging of malignant tumours. For example, endoscopic ultrasound-guided FNA of a paraoesophageal lymph node can detect metastatic oesophageal carcinoma. Another useful role for FNA is in diagnosing recurrence of malignancy, particularly lymphoma. Clinical FNA procedures are described and potential causes of an inadequate—i.e. non-diagnostic—aspirate are discussed. Various modalities of image guidance are discussed and methods of slide preparation compared. The role of special techniques such as immunocytochemistry and fluorescence in situ hybridization is illustrated by means of diagnostic scenarios. The uses and limitations of FNA cytology of the breast, thyroid, lymph nodes and salivary glands are discussed.
Aim The aim of FNA is to collect sufficient representative material from the lesion to enable a cytological (i.e. based on the appearance of dispersed individual cells and cell groups) diagnosis to be made. This contrasts with a histological diagnosis (i.e. based on the appearance of cells and their arrangement within tissue).
FNA versus tissue core biopsy (Table 1)
Keywords breast; FNA cytology; lymph node; pathology; thyroid
The use of FNA can obviate the need for tissue core biopsy or more invasive tissue sampling. This is particularly important in Biopsy is the removal of tissue from a living subject in order to establish the presence or extent of a disease. Sometimes, tissue may be taken to detect the cause of a disease (e.g. causative Helicobacter pylori may be identified in a biopsy from an area of
John R Mitchard MRCPath MPH is a Consultant Histopathologist and Cytopathologist at the Royal Cornwall Hospital Trust, Truro, UK. Conflicts of interest: none declared. Kathleen E Romain APCP CYTO is a Consultant Cytopathologist at Cheltenham General and Gloucestershire Royal Hospitals, Gloucestershire, UK. Conflicts of interest: none declared. Neil A Shepherd DM FRCPath is a Consultant Histopathologist at Gloucestershire Royal and Cheltenham General Hospitals, Gloucestershire, UK, and Visiting Professor at Cranfield University, London, UK. Conflicts of interest: none declared.
SURGERY 26:2
Figure 1 A fine needle passes horizontally into a mass lesion in the liver under CT guidance.
38
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
pneumothorax is not an infrequent occurrence following FNA of the lung. The risk of seeding tumour along the needle path appears to be very small.
FNA versus tissue core biopsy Feature
FNA
Tissue core biopsy
Speed of reporting
Results possible on same day Not needed (generally) Usually minor
In general, result obtained the following day May be needed May be serious
Common
Less common
Low Large numbers of cells Not preserved Can be done at bedside Not usually
Higher Smaller numbers of cells Preserved Difficult at time of biopsy Usually
Anaesthesia Complications of procedure Inadequate specimens Cost Cellular sampling Tissue architecture Assessment of adequacy of sample Spare tissue for special tests
Technique Operator Radiologists will perform most image-guided aspirates, but physicians, surgeons and cytopathologists may participate in the FNA of palpable lesions. Practice varies between centres in the UK. Some cytopathologists perform all clinical aspirates of breast and thyroid lumps, whereas all aspirates are performed by surgeons in other centres. All operators must be suitably trained and their performance monitored. Superficial aspirates In most situations, a 21-gauge (green) needle is attached to a 20 ml syringe, leaving 5 ml of ‘dead space’ in the syringe (which permits expulsion of the material onto the slide and eliminates the need to detach and reattach the needle in order to introduce air). The skin is cleaned with an alcohol swab and the lesion held between the thumb and index finger of the non-dominant hand. The tip of the needle is advanced into the lesion and 6–10 ml of suction is applied to the syringe (a syringe holder may be beneficial). Several passes are made through the lesion in various planes, the needle being rotated between each pass. The suction is released and the needle withdrawn when a drop of blood or fluid is seen in the needle hub. It is the capillary action of the hollow needle (rather than the negative pressure applied by the syringe) that is responsible for sampled material moving into the bore. It is sometimes preferable to use a needle without a syringe, especially in highly vascular sites like the thyroid gland. The role of suction is to empty the needle and allow necrotic material or cyst fluid to be removed from masses. The needle should be removed and firm pressure applied to the area if excessive bleeding is noted. It may then be possible to re-attempt the aspiration from a different angle. Otherwise, FNA can be performed one week later. According to the preferences of the reporting cytopathologists, the sample can be spread onto a slide, air dried and/or fixed or injected into transport medium. The causes of non-diagnostic aspirates are given in Table 2.
Table 1
advanced malignancy when a tissue diagnosis is needed to plan palliative management. FNA is a relatively inexpensive procedure compared to tissue core biopsy because less laboratory time and fewer reagents are consumed in preparing the slides. Cost minimization is very important because FNA is used widely in the evaluation of breast lesions detected as part of the UK Breast Screening Programme. Many centres in the UK operate ‘one-stop’ breast clinics (in which women with breast lumps are assessed clinically, radiologically and cytologically) because stained slides of FNA specimens can be prepared in a short time, enabling rapid diagnosis (often on the same day). FNA is a convenient, relatively painless and inexpensive procedure, but it must not be used indiscriminately. The aspiration of impalpable or non-existent lesions in clinic will frequently yield smears that are non-diagnostic or misleading, leading to a perception that FNA is a poor diagnostic tool. Clinicians must be aware of the limitations of cytological dia gnosis. For example, FNA cannot distinguish between: • in situ and invasive carcinoma of the breast • a follicular adenoma and follicular carcinoma of the thyroid gland (see below).
Risk
Possible causes for a non-diagnostic FNA
In general, FNA of superficial lesions is a safe procedure; bleeding is the main complication. Puncture of adjacent structures (e.g. the trachea in thyroid aspiration) occurs occasionally, but is usually straightforward to manage. Other risks include puncture of the jugular vein, carotid artery and apical pleura in the supraclavicular fossa. These may be avoided by detailed knowledge of anatomy and landmarks, and careful palpation of the lesion on examination of the patient. Aspiration of deeper structures carries risks associated with the path of the needle. For example,
SURGERY 26:2
• Lesion does not exist • Needle misses lesion • Aspiration of contents of cystic/necrotic lesion • Haemorrhage obscures diagnostic material • Low yield of cells from a sclerotic neoplasm • Inexperienced operator Table 2
39
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
The slides are air dried or fixed immediately with alcohol. Air-dried smears can be stained with May–Grunwald–Giemsa or Diff-Quik™, allowing rapid staining and assessment of the smear within minutes of the aspirate being taken. Thus, a cytopathologist can evaluate the smear, if adequate cellular material has been sampled, before the patient leaves the department. This is particularly important for deep-seated lesions, where an inadequate aspirate would entail another session in an already overstretched radiology department. May–Grunwald–Giemsa or Diff-Quik™ stains are the best for demonstrating cytoplasmic detail and stain colloid particularly well in thyroid aspirates. Papanicolaou stain is used most commonly for fixed slides and shows nuclear detail very clearly.
Deep/image-guided FNA Aspiration of deep-seated lesions is predominantly the domain of the radiologist and a detailed discussion is outside the scope of this contribution. The chosen modality will usually be that which enables the FNA to be performed most quickly because most radiology departments in the UK have huge demands placed on CT and MRI scanners. Ultrasound allows real-time guidance of the needle with imaging in any plane. Use is limited because ultrasound is not transmitted through air or bone. The needle is manipulated with one hand while the other hand holds the transducer so that the tip of the needle can be visualized. Ultrasound-guided FNA is a technically demanding procedure. Endoscopic ultrasound-guided fine-needle aspiration biopsy allows guidance of an aspiration needle placed endoscopically into the pancreas, a mediastinal lymph node or perigastric lymph node via the wall of the duodenum, oesophagus or stomach. Diagnosis of pancreatic masses and staging of gastric and oesophageal cancer is the most common indication for this investigation.
Indirect smears are usually prepared in the cytology laboratory. Following aspiration, needle washings are prepared at the bedside. Transport medium or fixative is aspirated via the attached needle into the syringe and washed back into the transport vial for centrifuging in the pathology department. This method offers the possibility of spare slides being available for special stains (including immunocytochemistry). May–Grunwald–Giemsa stains cannot be performed unless a separate FNA pass is performed to prepare an air-dried smear. Direct communication with the pathologist is recommended if the laboratory does not have clear guidelines for the preparation of FNAs.
Fluoroscopy is used for real-time guiding of a needle into lesions that are visible in two planes. The needle is inserted along the axis of a central X-ray beam and an orthogonal beam assesses the proximity of the tip of the needle to the lesion. Lung lesions (especially those in the lower zones) are usually amenable to FNA guided by fluoroscopy.
Special techniques in the laboratory The use of special laboratory techniques in FNA is reliant on cytology having adequate excess cellular material in the form of needle washings or multiple fixed slides prepared directly.
CT scanning allows sampling of small lesions almost anywhere in the body; the lung, liver (Figure 1) and retroperitoneum are the common sites. A vertical or horizontal trajectory is used, allowing the full length of the needle to be visualized (Figure 1). The passage of a fine needle through abdominal structures (e.g. small intestine) does not appear to cause problems. However, the colon should be avoided because of the risk of spreading bacteria along the needle track.
Immunocytochemistry is used widely in histopathology because abundant excess material is usually available. Material is often limited in FNA samples, so antibodies must be chosen carefully. The cytological appearances (along with the radiological and clinical findings) should be considered to produce a differential diagnosis. For example, FNA of a lung mass may reveal an adenocarcinoma. In a middle-aged female, this might represent a primary tumour of the lung or a metastasis from the breast. Immuno staining with antibodies to thyroid transcription factor-1 and oestrogen receptors may distinguish between the two. Diagnosis of metastatic melanoma or carcinoma and subtyping of malignant lymphoma are other instances where immunocytochemistry might be useful.
Slide preparation Following aspiration, most of the cellular material is within the needle bore. The material is expressed onto the glass slide using the residual 5 ml of dead space in the syringe after suction is released. Direct smears are the conventional method for preparation of aspirated material. A slide is labelled with the patient’s details and the needle contents gently expressed onto one end of the slide. A single layer of cells can be produced on the slide without crushing them by spreading the material with the needle or by using a second glass slide. The second glass slide can be held at an angle of about 30–40° to the first slide and the end brought into contact with the droplet (which is allowed to distribute along its edge). The second slide is then drawn along the first in a manner similar to that used to produce a blood film. Alternatively, the material can be gently ‘sandwiched’ between two slides that are then drawn along each other’s lengths to produce two smears; this method is suitable for ‘dry’ aspirates.
SURGERY 26:2
Fluorescence in situ hybridization can be applied to relatively small numbers of interphase cells, so it can be applied to FNA cytology. Fluorescence in situ hybridization can help to diagnose subtypes of non-Hodgkin’s lymphoma and sarcomas by detecting specific chromosomal translocations. HER-2/neu alterations in breast cancer aspirates can also be detected by this method.
Specific sites Breast Although the use of tissue core biopsy is increasing, FNA cyto logy is central in the diagnosis of breast cancer. 40
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
Thyroid In general, FNA is a first-line tissue investigation for solitary thyroid nodules and is also useful in the context of clinically and radiologically malignant tumours, when potentially treatable lymphomas can be distinguished from anaplastic carcinomas. All personnel involved in the taking and reporting of thyroid FNAs (and those acting upon the reports) must be aware of the limitations of the procedure. The patient is positioned supine with a pillow behind the shoulders (allows the neck to extend and the sternocleidomastoid muscles to separate). A 23-gauge (blue) needle should be used because the gland is highly vascular. For most lesions, three passes in a single plane usually offers the best compromise between sampling sufficient cellular material and causing excess contamination by blood. Some sclerotic tumours may need more passes. Local complications include haemorrhage, carotid puncture and tracheal puncture: the latter may cause coughing and haemoptysis (rare). Sampling without suction may be helpful for lesions where previous FNA has caused excessive blood contamination. The majority of solitary nodules will be dominant colloid nodules in a multinodular goitre or follicular adenomas. A minority will be follicular carcinomas, papillary carcinomas and other tumours (including anaplastic and medullary carcinomas). It is not possible to distinguish between follicular adenoma and carcinoma on cytological grounds because the distinction is based
FNA cytology scale of the breast C1: Inadequate (<5 groups of epithelial cells) C2: Benign C3: Atypia (probably benign) C4: Suspicious of malignancy C5: Malignant Table 3
Stereotactic FNA is commonly used for the sampling of small lesions. Mammograms are taken from two angles in order to calculate the coordinates of the lump. The position of the needle holder is set accordingly. FNA cytology of the breast is reported on a scale of C1 to C5 (Table 3). Benign lesions of the breast have cohesive groups of epithelial cells in a background of bare myoepithelial cell nuclei (Figure 2a), whereas the cells of malignant aspirates are dys cohesive and exhibit nuclear pleomorphism (Figure 2b and c). Clinical examination and radiological findings are similarly scored. A tissue core biopsy is performed if there is discrepancy between the three arms of the assessment. A tissue core biopsy has the advantage of: • providing sufficient material for hormone receptor studies • allowing distinction between in situ and invasive carcinoma.
FNA cytology of the breast a
b
c
a The smear shows the characteristic cytological features of a benign lesion. There is a large group of benign epithelial cells showing a ‘staghorn’ morphology; numerous bare/stripped nuclei (myoepithelial cells) are in the background. The lesion was a fibroadenoma and the cytology is distinctive for this diagnosis. b The smear is taken at the same magnification as a and shows a malignant lesion characterized by dyscohesive epithelial cells. c The smear is the same sample as b, but taken at a higher magnification to show the irregular pleomorphic nuclei in the epithelial cells. Figure 2
SURGERY 26:2
41
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.
Basic science
Correlation of FNA and histology in papillary carcinoma of the thyroid gland a
b
a FNA cytology shows enlarged thyroid epithelial cells with an obvious nuclear inclusion. b Subsequent histology shows a tumour composed of papillae (finger-like processes lined by epithelium with a fibrovascular core) lined by epithelial cells with pleomorphic, open nuclei.
Figure 3
on the histological presence (carcinoma) or absence (adenoma) of capsular and vascular invasion. The initial treatment for both neoplasms is lobectomy and so the FNA is generally reported as being consistent with a follicular lesion. It is often impossible to distinguish cytologically between a follicular neoplasm and a colloid nodule. Papillary carcinoma has classic nuclear features (Figure 3a and b) and treatment is total thyroidectomy. Attempts to formalize the reporting of FNAs of the thyroid into five categories (in a similar system to that used for the breast) have not met with universal acceptance.
nodes are not unusual in squamous cell carcinoma of the tonsil or posterior tongue; however, these aspirates may be misinterpreted as branchial cysts. The diagnosis of lymphoma by FNA allows planning of formal lymph node biopsy and staging procedures. Formal biopsy allows assessment of lymph node architecture and provides abundant material for immunocytochemical assessment. FNA can provide material for ancillary tests (e.g. flow cytometry, immunohistochemistry), but full categorization of the tumour at the time of primary diagnosis is desirable because it provides archive material for further tests, should new treatments become available. FNA is extremely useful for diagnosing recurrences.
Lymph node FNA of a lymph node is useful in the management of lymphadenopathy, enabling the diagnosis of reactive lymphadeno pathy and suggesting specific conditions (e.g. tuberculosis, toxoplasmosis); supplementary investigations (e.g. Mantoux testing, toxoplasma serology) can then be suggested. Excision biopsy can be undertaken if no cause is found and lymphadenopathy persists. The diagnosis of metastatic malignancy is important to recognize because disseminated disease is often actively managed. The appearance of the tumour cells may suggest a primary site. Necrotic metastases in cervical lymph
SURGERY 26:2
Salivary glands The major salivary glands are readily accessible with a needle and FNA of masses is usually attempted. Pleomorphic adenoma is the most common tumour of the major glands. Determining whether a mass is non-neoplastic (e.g. a focus of chronic sialadenitis), benign or malignant can influence the nature of the definitive surgery (or whether surgery takes place). For example, Warthin’s tumour (adenolymphoma) is a benign tumour most often seen in the elderly and surgery may not be necessary. ◆
42
Crown Copyright © 2007 Published by Elsevier Ltd. All rights reserved.