Fine needle aspiration cytology (FNAC) is now a popular and effective technique to have a rapid tissue diagnosis. It is a simple, cheap and rapid diagnostic procedure (Box 1.1).1
There is loss of complex architecture in FNAC smear. So at times, it is difficult to categorize the lesion exactly particularly in soft tissue or skin lesion. In situ carcinomas are also difficult to diagnose in FNAC. Similarly, the capsular invasion or lymphovascular emboli cannot be detected in FNAC. Interpretation of FNAC smear needs considerable training and practice.2 The diagnostic accuracy of FNAC largely depends on the interpreter's experience in this field (Box 1.2).
FNAC is usually free of significant complications. There may be bleeding, hematoma, emphysema (in lung FNAC). Rarely anaphylactic reaction may occur due to accidental rupture of hydatid cyst during FNAC procedure. However, these complications can be avoided if proper precautions are taken during this procedure. The myth of spillage of tumor through the needle tract is not true if fine needle is used.3 In Postgraduate Institute of Medical Education and Research, Chandigarh, India, we have been doing FNAC for more than 40 years and we did not encounter a single incidence of seeding of tumor cells by the needle tract.
FINE NEEDLE ASPIRATION TECHNIQUE
It is always preferable that cytopathologists himself/herself should perform FNAC.4,5 We routinely use 22 to 23 gauge needle, 20 ml syringe and a pistol handle for doing FNAC (Figure 1.1A). The needle is connected to the plastic syringe and the syringe is properly fitted with the pistol handle (Figure 1.1B). The handle gives support and also helps to apply suction during the procedure.
A thorough clinical history and physical examination is mandatory prior to the technique. This will give the initial impression of the disease. The patient also should be reassured properly and should be explained briefly about the procedure.
Next step is to clean the area by a clean spirit swab (Figure 1.2A). The swelling to be aspirated is held in between the two fingers to make it immobilized. The needle is swiftly introduced within the mass. The direction of the needle should be perpendicular to the swelling. The needle should be moved to and fro within the lesion. The repeated movement of the needle cuts the tissue and this is mandatory for getting adequate loose cut out tissue. Simultaneously, the plunger is also retracted to create a negative pressure (Figure 1.2B). The majority of times, the material remains in the needle hub. The needle is withdrawn sharply along with the release of the plunger to stop the suction. Releasing the negative pressure before withdrawing the needle is a vital step to get adequate material.
Figure 1.1A: Essential instruments needed for fine needle aspiration cytology are displayed such as pistol handle, syringe, needle and glass slide from left to right
The needle is then rapidly detached from the syringe and the plunger is retracted to get enough air within the syringe. The needle is reattached and the air is pushed to eject the material on the glass slide (Figure 1.2C). The smear is made by gently pressing one clean slide over the other and moving the upper slide over the lower one to spread the material (Figure 1.2D). Multiple smears are made and both air dried and alcohol fixed smears should be kept for staining.
Figure 1.2B: FNAC procedure: Needle is introduced in the swelling and is gently moving to and fro. Simultaneously negative suction is also created by withdrawing the piston
Figure 1.2D: FNAC procedure: The aspirated material is expelled and the smear is made by gently pressing the upper slide on the lower one
The residual material should be rinsed in citrate buffer solution and can be processed for ancillary techniques. A repeat FNAC could be done to get more material for the ancillary techniques.
In case of deep seated lesions, the whole procedure should be performed with the direct help of an experienced radiologist. FNAC can be done under the guidance of ultrasonograph (USG), computerized tomography (CT) or fluoroscopy. Long thin needle is used for FNAC of deep seated lesions. Local anesthesia is required for the aspiration biopsy. However, no hospitalization is required. An immediate rapid Giemsa stain could be done to evaluate the adequacy of the sample.
Fine needle sampling: Non-suction fine needle sampling (FNS) is a helpful technique in certain situations like small lesion, thyroid swelling or breast lesions.6 In this technique, the swelling or the area to be aspirated should be pressed in between the two fingers and a 23 gauze needle is gently introduced (Figure 1.3A). The needle is moved to and fro in different directions (Figure 1.3B). The material comes to the needle hub by capillary suction. The needle is gently withdrawn and the syringe filled with air is attached with the needle hub. The aspirated material is expelled gently on the slide by moving the piston of the syringe (Figure 1.3C). The major advantage of FNS is to get abundant material in a bloodless background (Box 1.3). FNS is particularly important in vascular organ like thyroid.
Figure 1.3A: FNS procedure: The swelling is held in betweenthe two fingers and the needle is introduced vertically
Figure 1.3B: FNS procedure: The needle is gently moved to and fro within the swelling. The material comes to the hub of the needle by capillary action
Figure 1.3C: FNS procedure: The needle is reattached with the syringe filled with air and the material is expelled on the glass slide
EVALUATION OF FNAC SMEAR
FNAC smear of each case should always be interpreted along with clinical history. In case of any doubt about the cytological diagnosis, the smears should be reviewed in the light of clinical findings. If necessary, a repeat FNAC could be done or the surgical biopsy should be performed. For the initial evaluation of FNAC smear, the cytopathologist should always have the checklist of the important features such as cell arrangement, cell morphology and background materials (Box 1.4).
Cellularity: It is important to assess the overall cellularity of the smear. The cellularity of the sample varies widely depending on the skill of the individual cytopathologist. In general, FNAC of the malignant lesions yield good amount of cells because of loss of cell cohesion. However, some malignant lesions may have low cellular yield due to sclerosis or other degenerative changes.
Cell population: The population of cells on the smear gives important information. What are the different varieties of cells present on the smear? Are they monomorphic or polymorphic? Are the benign cells admixed with the neoplastic cells? In lymph node, monomorphic population of lymphoid cells indicates the possibility of non-Hodgkin's lymphoma. Hepatocellular carcinoma often shows dysplastic liver cells along with frank malignant cells. Benign hepatocytes are often noted along with the malignant cells in case of metastatic malignancy in liver. Similarly, bipolar myoepithelial cells are noted in benign breast lesions. The associated cells provide lot of information about the nature of the disease.
Cell arrangement (Figures 1.4A to F): The complex tissue architecture is lost in FNAC smear. However, the cell architecture is still predictable in FNAC. There are different architectural pattern in FNAC which can be recognized with confidence (Box 1.5).
The study of the detailed cytomorphology of the individual cells is very important for accurate diagnosis.
The morphologic changes of the nucleus are the reflection of the pathology of the cell. The nuclear size, shape, pleomorphism, margin, nucleoli, chromasia and chromatin pattern should be noted carefully (Figure 1.5). The general criteria of malignancy have been mentioned in Box 1.6.
It is important to note that the nuclear changes of the cell may not be the sole criteria of malignancy. The diagnosis of malignancy largely depends on the aspiration sites and clinical history. For example, nuclear enlargement and pleomorphism may be remarkable in many benign tumors such as pleomorphic lipoma, paraganglioma, angiomyolipoma, or ancient schwannoma.
Figures 1.4A to F: Cellular arrangement: (A) Gland like arrangement in adenocarcinoma (MGG X 440); (B) Central ball like pinkish material surrounded by cells in adenoid cystic carcinoma (MGG X 440); (C) Rosette with central fibrillar material surrounded by cells in neuroblastoma (H & E X 440); (D) Papilla in a case of papillary tumor of breast (H & E X 440); (E) Row of cells as trabecular pattern in hepatocellular carcinoma (MGG X 440); (F) Fascicular arrangement of cells in a sarcoma (H & E X 440)
On the other hand, the reverse is also true. Many malignant tumors may have deceptively monomorphic nuclei such as mucinous tumor of the breast, adenoid cystic carcinoma, acinic cell carcinoma or follicular carcinoma of thyroid.
Abnormal chromatin pattern is possibly one of the most important criteria of malignancy. Irregular clumped chromatin is characteristically seen in malignant cells. The other types of chromatin patterns are fine reticular, salt and pepper and cart wheel appearance (Figures 1.6A to D).
Figures 1.6A to D: Different types of chromatin pattern: (A) Irregular coarse clumped chromatin (PAP X 1200); (B) Salt and pepper chromatin in neuroblastoma (H & E X 1200); (C) Fine opened up chromatin in leukemic blast (MGG X 1200); (D) Peripheral clumped chromatin (cart wheel appearance) in plasma cell (H & E X 1200)
The morphological appearances of the nucleus and their significance have been mentioned in Box 1.7.
The cytoplasm of the individual cells also provides valuable information on cell function and cell type. One should carefully note about the volume of the cytoplasm, tinctorial character, vacuolation, cytoplasmic granularity, intracytoplasmic lumina, cytoplasmic inclusions and any other changes (Box 1.8 and Figures 1.7A to D).
Cytoplasmic granularity of the cell is better evident in MGG stain. Intracytoplasmic keratin is better demonstrated in Papanicolaou's stain. Lipid in cytoplasm of the renal cell carcinoma can be shown by oil red O stain (Box 1.9).
BACKGROUND ACELLULAR MATERIALS9
Background of the FNAC smear may provide useful diagnostic information (Box 1.10 and Figures 1.8A to F). Lace like vacuolated background of the smear is known as tigroid background. Tigroid background is usually seen in seminoma/dysgerminomas. It may also be present in rhabdomyosarcoma and adrenal adenoma. Mucinous material in the background is seen in mucinous adenocarcinoma or any other mucinous cyst. Fatty material is noted in lipomatous neoplasm. Fat necrosis also shows degenerated fat. Colloid is seen in colloid goiter. Concentric laminated calcification, known as Psammoma bodies, is seen in papillary carcinoma of thyroid, meningioma and epithelial carcinoma of ovaries.
Figures 1.7A to D: Cytoplasmic changes: (A) Large vacuoles in the cytoplasm in a case of adenocarcinoma (MGG X 1200); (B) Cytoplasmic reddish granules in a neuroendocrine tumor (MGG X 1200); (C) Dark blotchy black pigment in the cytoplasm in a case of malignant melanoma (MGG X 1200); (D) Black carbon pigment in the bronchial lining cells in lung FNAC material (PAPX 1200)
Figures 1.8A to F: Different types of background materials: (A) Light blue background due to colloid (MGG X 440); (B) Chondromyxoid background in a case of chordoma (MGG X 400); (C) Mucoid background in mucinous adenocarcinoma; (D) Chondroid background in a chondrosarcoma (MGG X 440); (E) Light blue necrotic background in a case of tuberculosis (MGG X 440); (F) Mossy background of synovial fluid in case of synovial cyst (H & E X 440)
Amorphous eosinophilic hyaline globules are noted in collagenous spherulosis of breast. Various crystals may also be seen in different lesions such as Reinke's crystal in sertoli cell tumor of testis, tyrosine crystals in pleomorphic adenoma of the salivary gland and sulfur crystals in prostatic carcinomas.
Final reporting: Final reporting of the FNAC smear should always be given on the pre-context of clinical information. In many centers major surgical operation is done or preoperative chemotherapy is administered on the basis of FNAC report. Adequate care should be taken before offering a definitive diagnosis. If there is any suspicion, either a repeat FNAC or surgical biopsy should be advised. A repeat FNAC from the multiple sites may yield a more representative material for diagnostic interpretation.
There are many potential mimickers of malignant cells and unless adequate care is taken, a false positive diagnosis may be given (Figures 1.9A to D).
- Reactive changes (Figure 1.9A): Reactive atypia noted in reactive mesothelial cells in intra-abdominal FNAC is a potential mimicker of malignancy. The window pattern in between the cells is a helpful feature to identify the mesothelial cells. Reactive atypia is also noted in bronchial epithelial cells, benign lesions of breast and liver FNAC around cirrhotic nodules.
- Nuclear pleomorphism (Figure 1.9B): Marked nuclear pleomorphism is often noted in paraganglioma, adrenal adenoma and ancient schwannoma. A careful clinical history and radiological localization are helpful to avoid a false positive diagnosis in these lesions.
- Endometriosis (Figure 1.9C): The presence of endometrial glands in the subcutaneous tissue in case of endometriosis may be misinterpreted as metastatic adenocarcinoma. The gland in endometriosis is usually monomorphic. The clinical history is also characteristic. The patient complains of pain and enlargement of the swelling during menstruation.
- Radiation effect: Radiation or chemotherapy may cause nuclear enlargement. The nuclear texture is not well preserved in these cases and this is a helpful clue along with clinical history.
- Muscle degeneration: Muscle trauma and inflammation may give rise to large bizarre looking cells. The cytoplasm of this cell is abundant and cross striations may be noted.
- Granulomas: Ill formed granulomas may show spindle looking cells and may be mistaken as spindle cell neoplasm.
- Viral infections (Figure 1.9D): Large atypical mononuclear cells in the viral infection may be mistaken as immature lymphoid cells and a false diagnosis of lymphoma may be given.
Immunocytochemistry: Immunocytochemistry is essential for the accurate diagnosis of many lesions.
Figures 1.9A to D: Mimickers of malignancy: (A) Reactive mesothelial cells may often mimic as malignant cell (H & E X 440); (B) Marked nuclear atypia in paraganglioma (H & E X 440); (C) Endometriosis may often mimic as carcinoma (H & E X 240); (D) Atypical lymphoid cells in viral infection (H & E X 440)
It is particularly helpful in diagnosis of poorly differentiated malignancies, diagnosis and subtyping of lymphomas, categorization of malignant round cell tumor, differentiating reactive mesothelial cells from malignancy and also diagnosis of many sarcomas (Box 1.11).
The diagnostic utilities of various antibodies are mentioned in Table 1.1. Indiscriminate use of immunocytochemistry should not be done. After the initial assessment of the FNAC smear, a working panel of antibody should be applied, preferentially on the cell block preparation.
- Brown LA, Coghill SB. Cost effectiveness of a fine needle aspiration clinic. Cytopathology 1992;3(5):275–80.
- Orell SR. Pitfalls in fine needle aspiration cytology. Cytopathology 200;14(4):173–82.
- Lundstedt C, Stridbeck H, Andersson R, Tranberg KG, Andrén-Sandberg A. Tumor seeding occurring after fine-needle biopsy of abdominal malignancies. Acta Radiol 1991;32(6):518–20.
- Coghill SB, Brown LA. Why pathologists should take needle aspiration specimens. Cytopathology 1995;6(1):1–4.
- Polacarz SV. Why pathologists should take needle aspirates. Cytopathology 1995;6(5):358
- Dey P, Ray R. Comparison of fine needle sampling by capillary action and fine needle aspiration. Cytopathology 1993;4:299–303.
- Dey P. Cancer nucleus: morphology and beyond. Diagn Cytopathol 2010;38(5):382–90.
- Dey P. Chromatin pattern alteration in malignant cells: an enigma. Diagn Cytopathol 2005;32(1):25–30.
- Pattari SK, Dey P. Facts about artefacts in diagnostic pathology. Indian J Pathol Microbiol 2002;45(1):133–5.