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Fine Needle Aspiration Cytology

Presented by:Dr Kalpajyoti Bhattacharjee


INTRODUCTIONFine Needle Aspiration Cytology (FNAC) is a technique whereby cells are obtained from a lesion using a thin bore needle and smears are made for cytopathological diagnosis.This technique is based on the fact that tumor cells are less cohesive and are easily aspirated.Used in the diagnosis of breast lumps, thyroid nodules, liver disease, subcutaneous soft tissue mass, salivary gland diseases and oral diseases.

Oral cavity is a site where mucosa is very vascular and an open biopsy leads to a lot of bleeding which is difficult to control.In recent times FNAC solves these problems, adequate material can easily be obtained by using a 10 ml. syringe from an intraoral or extraoral site without any discomfort to the patient and with no bleeding.In some cases a subsequent surgery is not needed and patient can be put on appropriate treatment.FNAC report is prepared within 24 hours of sampling gives early, quick information to the surgeon about the type of lesion he is dealing with.

HISTORICAL PERSPECTIVEThe technique was introduced in the 1930s by Martin and Ellis in the United States, but it never became widespread. Since the 1950s it has been used extensively in Scandinavia and in Holland.Fine Needle for aspiration were first introduced in Europe in the 1950s by Lopez-Cardozo in the Netherlands and Soderstrom in SwedenPublication by Zajicek from Karolinska Hospital in Stockholm that brought aspiration cytology to international alterations.

ADVANTAGESimple office technique Rapid diagnosis Economical Sampling from multiple sites in the same sitting High diagnostic accuracy Many techniques such as bacterial culture, immunocytochemistry, flow cytometry, cytogenetics, polymerase chain reaction, etc. are possible from FNAC material.

LIMITATIONS Loss of tissue architecture Capsular invasion and lymphovascular invasions cannot be detected Difficult to differentiate in situ versus invasive carcinoma Considerable training is needed for accurate interpretation.

FNAC AS A TOOL IN CLINICAL INVESTIGATIONInitially used as a mean to confirm a clinical suspicion of local recurrence or metastasis of known cancer without subjecting the patient to further surgical intervention.Inflammation, infection, degenerative conditions, in diagnosis and monitoring of graft rejection in transplantation surgeryAlternative or complement to frozen sectionIntraoperative cytology

THE PRACTICE OF FNACSuccess of FNAC depends on four fundamental requirement:Samples must be representative of the lesion investigated.Samples must be adequate in terms of cells & other tissue componentsSamples must be correctly smeared and processed Biopsy must be accompanied by relevant and correct clinical/radiological information.

EQUIPMENTSNEEDLES: Routinely 22-23 gauge needle used.Syringes: 20 ml syringePistol handle

Sterile container: Physiological saline or Hanks balanced solutionSlides: clean, dry & free of grease.A 0.4 mm haemocytometer coverslip gives better control over smearing pressure & a more perfect spreadFixatives: 70-90% ethanol, Carnoys fixative, 10% buffered formalin, gluteraldehyde usedStainsMicroscopes

FINE NEEDLE ASPIRATION TECHNIQUE 2 techniques:FNAC with aspirationFNAC without aspiration

Site of FNAC should be cleaned by spirit swab

Needle is introduced in the swelling and is gently moving to and fro. Simultaneously negative suction is also created by withdrawing the piston

Air is taken in the syringe and needle is reattached

The aspirated material is expelled and the smear is made by gently pressing the upper slide on the lower one

FNAC with aspiration

-Introduced by Zajdela in 1987-based on the observation that the capillary pressure in a fine needle is sufficient to keep the detached cell inside the lumen of the needleFNAC without aspiration

FAILURE TO OBTAIN A REPRESENTATIVE SAMPLENeedle has missed the target tangentiallyNeedle in central cystic/necrotic/hemorrhagic area devoid of diagnostic cells.Needle in dominant benign mass missing a small adjacent malignant lesions.Fibrotic/desmoplastic target tissue giving a scant cell yield.

PROCESSING THE SAMPLE Sample expelled on to a clean & dry microscope slide using air in a syringe.

Indirect smearing: Thin fluid samples are best processed by centrifugation on the cytocentrifuge.Milipore nucleopore filtration is an alternativeThinprep technique

FIXATION & STAINING2 fundamentally different methods of fixation & staining are used in FNAC:Air drying followed by staining with a haematological stain such as MAY GRUNWALD- GIEMSA STAIN , Jenner-Giesma, Diff-QuikAlcohol fixation and staining according to PAP or with H&E.

Papanicolaou stainContents:Harriss hematoxylinOrange G6EA 50Results:Nuclei- blue/blackCytoplasm (non-keratinizing)- blue/greenKeratinizing cells- pink/orange

Romanowsky stainContents: Methylene blue/azure B and eosin, dissoved in acetone-free methanol, include jenner, Giesma, May Grunwald and Leishman stain

Results:Nuclei- purple/blueCytoplasm- pink/blueEosinophils- pink/red

Diff-Quikis a commercialRomanowsky stainvariant, commonly used inhistological stainingto rapidly stain and differentiate a variety of smears, commonly blood and non-gynecological smears, including those of fine needle aspirates.

MAY GRUNWALD- GIEMSA STAINcommonly used staining of blood smearsContents:methylene blue (a basic dye)Azures (also basic dyes)Eosin (an acid dye)

Results: Nuclei of white blood cells and the granules of basophil granulocytes- blueRed blood cells and eosinophil granules redcytoplasm of white blood cells - light blue

SPECIAL STAINSPAS or Alcian blue - mucins, glycogen Prussian blue - ironMasson-Fontana - melaninCongo red - amyloidZiehl-neelson - acid fast bacilliBile pigment- Fauchets reagent counterstained with sirus red.Gram, PAS or Gomoris silver stain for microorganism

COMPLICATIONSUsually free of complicationsBleedings, hematoma, emphysema (in lung).Rarely anaphylactic reaction- accidental rupture of hydrated cyst

FNAC OF SALIVARY GLANDFNAC of the salivary gland lesions has gained wide clinical recognition. The incisional biopsy of the salivary gland may cause fistula formation and other complications that can be avoided in FNAC.For accurate diagnosis of salivary gland lesions, an adequate sample stained by both May Grunwald- Giemsa stain and Papanicolaous stain (or H and E) along with detailed clinical history are needed.

Cytology of Normal Salivary Glands Benign ductal cells: These cells are usually in small clusters or monolayered sheets. The cells are round to oval, with scanty cytoplasm having monomorphic nuclei.

Acinar cells: These are commonly present as small ball like clusters. The individual cells are round with abundant foamy cytoplasm and small round nuclei. The acinar cells may also be present discretely and cells with bare nuclei may be mistaken as lymphocytes.

Myoepithelial cells: These are oval to spindle shaped cells present near the basement membrane of the ductules. Oval plasmacytoid myoepithelial cells may also be seen.

Fibrous tissue: Fragments of fibrous and adipose tissue may also be seen in the background of the normal salivary aspirate.

Aspirate of normal salivary gland tissue showing grapelike clusters of epithelial cells composed of spherical acini and branching ducts (Romanowskys stain).

Benign acinar cells arranged in rosette formation. The cells have abundant foamy vacuolated cytoplasm with indistinct cell borders, and eccentric nuclei (Papanicolaou stain).

Diagnostic approach of salivary gland tumors

Fatty InfiltrationPresents as a diffuse enlargementAddition to normal salivary gland elements, there is significant increase in the amount of adipose tissue situated between ductal and acinar cellsAssociated with diabetes, cirrhosis, alcoholism, medications, nutritional deficiencies and hormonal disturbances

Chronic sialadenitis

Commonly results from stones or postsurgical scarringMore frequently seen in the submandibular glandCytology:low cellularity and show predominance of ductal cells, background has variable number of lymphocytes, occasional plasma cells and neutrophils, and spindle fibroblastsSquamous and mucinous metaplasia may be focally encounteredAcute sialadenitis: seldom sampled, many neutrophils are admixed with benign salivary gland elements, reactive and reparative changes may be seen

Short tubular segments of ductal epithelium composed of small hyperchromatic cells characteristic of chronic sialadenitis (Romanowskys stain).

Benign Lymphoepithelial Cysts of the Parotid GlandsCytology:Smears - characterized by a mixed lymphoid infiltrate with a predominance of small mature lymphocytesCharacteristically has a watery proteinaceous background in which the lymphoid elements are distributedCuboidal or mucin-containing cells may be found distributed singly in the smear

Aspirates of benign lymphoepithelial cysts are characterized by a mixed population of lymphoid cells dispersed in a watery background (Romanowskys stain).

Pleomorphic AdenomaPleomorphic adenoma (PA) commonly involves the parotid gland (more than 75%).painless, slow growing, firm to h