FINE NEEDLE ASPIRATION CYTOLOGY TECHNIQUE

An overview of the techniques for FNAC. To strength of a building always lies in its foundation. And today we will talk about the building brick of an ever high rising skyscraper of cytology, i.e. FNAC techniques.
The full form of FNAC is Fine Needle Aspiration Cytology.
It is a diagnostic technique by which we obtain cells from a lesion with a fine needle of less thickness which is usually around 22-25 G depending on the nature of the lesion.

1. FNAC TECHNIQUE Dr Atreyee Chakrabarty Junior Resident Department of Pathology, IMS, BHU Moderator: Dr Mahima Yadav

2. OUTLINE Introduction Components of a successful FNAC Advantages Disadvantages Contraindications Complications Equipments

3. Patient preparation Examination of lesion FNAC methods FNAC of specialised organ systems Recent advances References

4. INTRODUCTION FNAC=“Fine Needle Aspiration Cytology” Diagnostic technique Cells are obtained from lesion using fine bore needle. Smears are prepared and sent for cytopathological examination. First introduced in Sweden but popularized by Lopez Cardozo and Soderstrom .

5. COMPONENTS OF A SUCCESFUL FNAC Samples must be representative of the lesion being investigated. Samples must be adequate in terms of cells and other tissue components. Samples must be correctly smeared and processed . Correlation with clinical findings, radiological findings and histopathological examination.

6. ADVANTAGES Simple Rapid diagnosis Cost effective Multiple sites can be sampled in the same sitting. Material obtained can be used for: Immunocytochemistry Microbiological examination and culture Molecular and cytogenetic study

7. DISADVANTAGES Loss of tissue architecture No information about capsular and vascular invasion. Insitu vs invasive carcinoma cannot be differentiated. Personnel should be well trained to perform technique and interpret findings.

8. CONTRAINDICATIONS Uncooperative patient No valid consent Coagulation disorders and severe thrombocytopenia Pulsatile neck masses due to vascular etiology (e.g., cervical aortic arch [CAA], high riding brachiocephalic artery, vascular malformations, etc .) Infections , such as hydatid cysts Infected skin overlying the mass

9. COMPLICATIONS Usually free of complications Hematoma Infections Infarction of lymph node or thyroid Surgical emphysema Biliary peritonitis and bowel perforation Accidental rupture of aneurysmal vessel or spleen Hydatid cyst rupture- anaphylaxis Seeding of tumor through needle

10. EQUIPMENTS REQUIRED

11. NEEDLE Standard disposable needles SUPERFICIAL LESIONS DEEP LESIONS 23-27 G needle Large bore needle: hard and fibrotic lesion, cyst with viscous material, material for ancillary tests Small bore needle: small lesions, vascular tissues, children, sensitive sites. 22 G lumbar puncture needle with trocar. 22 G Chiba needle Franzen needle: pelvic lesions

12. Standard disposable plastic syringes 10-20 cc Syringe mounted on metal piston. Piston leaves one hand free to feel and fix target. Better precision while placing needle. Better suction. SYRINGES AND HOLDER

13. CONTAINERS Small sterile containers Tightly capped 95% ethanol: in Koplin jar for Papanicolaou’s stain Rinsing of needles and syringes 10% formalin: cell block for immunocytochemistry Physiological saline or Hank’s balanced salt solution: transport media for flow cytometry

14. SLIDES Slides should be clean, dry and free from grease. Frosted slides: Convenient for labelling . Coated slides: Convenient for immunostaining .

15. FIXATIVES STAINS MICROSCOPE

16. OTHER EQUIPMENTS Skin disinfectants Sterile dressings Local anaesthetic Tongue depressors Pocket torch Sterile scalpel blades Latex gloves Face masks

17. PATIENT PREPARATION Consent and clinical details: Informed consent and history along with clinical findings and radiological details. Supine position with easy access on either side. Couch with stirrups : Transrectal and transvaginal procedures . Examination chair with adjustable headrest: for head and neck lesions.

18. EXAMINATION OF A PALPABLE LESION

19. HISTORY Duration of the lesion Mode of onset Other associated findings Progress of the swelling Presence of lump in other sites Impairment of function History of recurrence Past history Personal history Family history

20. PHYSICAL EXAMINATION

21. ANAESTHESIA Pre biopsy sedation is rarely necessary. Local anaesthesia not required in superficial sites . Recommended in transpleural , transperitoneal and transperiosteal aspirations . Atropine: prevent vasovagal reflex in transpleural aspiration. Spray anaesthetic may be given for mouth, pharynx and other mucosal sites. For children, local anaesthetic ointment may be applied 30 mins prior.

22. FNAC OF SUPERFICIAL LESIONS

23. TECHNIQUES Fine needle aspiration Fine needle sampling

24. FINE NEEDLE ASPIRATION

25. NEEDLE INSERTION Near vertical needle insertion: Less painful Better appreciation of depth Near tangential needle insertion: Superficial skin lesions Chest wall lesions

26. FINE NEEDLE SAMPLING

27. Introduced by Zajdela in 1987 Principle: Capillary pressure in a fine needle is sufficient to keep the detached cells in the lumen. INDICATIONS ADVANTAGES DISADVANTAGES Vascular tissues Small mobile lymph nodes Soft and mobile lesions Less painful Less chances of admixture of blood Easy manipulation of needle Less material compared to fine needle aspiration. Unsuitable for cystic lesions and hard fibrotic lesions.

28. Creamy consistency High cellularity No blood or fluid Blood or fluid Less cellularity

29. DIRECT SMEARING

30. SMEARING OF DRY ASPIRATE

31. SMEARING OF WET ASPIRATE

32. INDIRECT SMEAR Thin watery samples are processed by cytocentrifuge = cytospin . Milipore or nucleopore can also be used. Needles or synriges can be rinsed in saline or formalin and then centrifuged and filtered on the slides. Thin Prep method is used for gynaecological specimens. Direct smear is preferred over indirect.

33. FIXATION AND STAINING

34. FIXATION - For May Grunwald Giemsa staining. - For Papanicolaou staining. - Done using 95% ethanol, isopropyl alcohol and methanol.

35. FEATURES AIR DRIED SMEAR WET FIXED SMEAR Stains Romanowsky stains Papanicolaou stains Dry smear Good fixation Drying artefacts common Wet smear Artefacts common Good fixation Tissue fragments Cells poorly seen Individual cells well visualized Cell and nuclear size Exaggerated , differences enhanced. Comparable to tissue sections Cytoplasmic detail Well demonstrated Poorly demonstrated Nuclear detail Not well demonstrated Excellently demonstrated Nucleoli Not always discernible Well demonstrated Stromal components Well shown and often differentially stained Poorly demonstrated Partially necrotic tissue Poor definition of cell details Individual cells well defined

36. TISSUE FEATURES PROMINENT ON GIEMSA STAIN FEATURES PROMINENT ON PAP STAIN EPITHELIAL TISSUE MUCIN: INTRACELLULAR OR EXTRACELLULAR COLLOID: THYROID SECRETORY GRANULES: PROSTATE LIPOFUSCIN GRANULES: SEMINAL VESICLE LIPID VACUOLES BARE BIPOLAR NUCLEI BILE PLUGS STROMAL GLOBULES AMYLOID SQUAMOUS DIFFERENTIATION/KERATINISATION ONCOCYTES NUCLEAR CHROMATIN PATTERNS NUCLEOLI NUCLEAR GROOVES LYMPHOID TISSUE CYTOPLASMIC BASOPHILIA LYMPHOID GLOBULES (LYMPHOGLANDULAR BODIES) HEMOPOEITIC CELLS LIPID VACUOLES NUCLEAR OUTLINE NUCLEAR CHROMATIN PATTERN NUCLEOLI

37. TISSUE FEATURES PROMINENT ON GIEMSA STAIN FEATURES PROMINENT ON PAP STAIN MESENCHYMAL TISSUES FIBROMYXOID/CHONDROMYXOID GROUND SUBSTANCE OSTEOID BASEMENT MEMBRANE AMYLOID INTRACYTOPLASMIC LIPID VACUOLES NUCLEAR DETAIL IN SOLID TISSUE FRAGMENTS NEUROENDOCRINE TISSUES CYTOPLASMIC GRANULARITY SPECKLED NUCLEAR CHROMATIN INFLAMMATORY TISSUE EOSINOPHILS MACROPHAGES

38. MUCIN COLLOID BARE BIPOLAR NUCLEI STRUCTURES VISUALISED BY MAY GRUNWALD GIEMSA STAIN HYALINE STROMAL GLOBULES

39. AMYLOID LYMPHOGLANDULAR BODIES CHONDROMYXOID STROMA STRUCTURES VISUALISED BY MAY GRUNWALD GIEMSA STAIN

40. KERATINISED SQUAMOUS CELLS ONCOCYTES NUCLEAR GROOVING NUCLEAR CHROMATIN STRUCTURES VISUALISED BY PAPANICOLAOU STAIN

41. SPECIAL STAINS STAINS STRUCTURE PAS, diastase, Alcian blue Mucin Prussian blue Iron Masson Fontana Melanin Grimelius Argyrophilic granules Congo red Amyloid Gram, PAS, Ziehl Neelsen , Gomori’s silver stain Microorganisms PAS Glycogen Fat Oil red O Fouchet reagent with Sirius red Bile pigment

42. ANCILLARY TECHNIQUES All ancillary techniques can be done for FNAC sample. Cell block: Immunocytochemistry Flow cytometry : Immunophenotyping . Electron microscopy used in some cases.

43. FAILURE TO OBTAIN REPRESENTATIVE SAMPLE Needle has missed the target tangentially. Needle has hit a cystic/necrotic/hemorrhagic area and missed the diagnostic area. Needle has hit the dominant benign mass and missed a small area of adjacent malignancy. Fibrotic/ desmoplastic target tissue gives scant cellularity.

44. FNA FOR DEEP SEATED LESIONS USG guided FNAC CT guided FNAC

45. USG GUIDED FNAC Gives optimal depth of biopsy Guides needle to the solid part of a lesion with solid and cystic components. Shows relationship of needle to other anatomical structures such as major vessels, pleura. No radiation exposure. However, there is significant obscuration due to air or bone.

46. CT GUIDED FNAC ADVANTAGES DISADVANTAGES High resolution Costly Exact localization of the needle Time taking procedure Image of traversing needle visible Risk of radiation exposure

47. FNAC OF SPECIALISED ORGAN SYSTEMS

48. HEAD AND NECK LESIONS

49. ORBIT Supervision by ophthalmic surgeon is recommended. Superficial lesions can be aspirated without image guidance. USG or CT guidance advisable for deep seated lesions. Patient made to lie in supine position and eye is fixed. Needle should be inserted close to the bony wall of the orbit. Risks: Retrobulbar hemorrhage, hematoma, infections, globe perforation and intracranial injury.

50. SALIVARY GLAND Primary method of evaluation of space occupying lesions Can distinguish between non neoplastic vs neoplastic and benign vs malignant . Preferred over incisional biopsy as: Potential risk of fistula formation Risk of seeding of tumor cells 23 G needle is used. Multiple sites should be aspirated. Repeat FNAC from cystic lesions after draining cystic contents.

51. THYROID LESIONS These lesions move with deglutition . Use of larger caliber needles not recommended . More than one aspiration per nodule is not necessary. Patient is placed in Rose’s position. Patient instructed to refrain from swallowing and speaking , only asked to continue breathing. Rapid needling and aspiration must be done except for cyst fluid. Side effects : pain, swelling, hematoma, infection, infarction, seeding of tumor cells.

52. Superficial Immobilised with one hand and aspirated using dominant hand Done under visualization using ultrasound guidance

53. THORAX

54. Can be solid or cystic. Detected by mammography, ultrasonography or MRI. Stahl suggested asking the patient to indicate the location of the lesion is more reliable than actual palpation. Two to four passes of the needle required. Ancillary examination can be done. Steroid receptor quantification : ER, PR Proliferation antigen : Ki67 DNA ploidy analysis and detection of gene expression : Her2

55. MEDIASTINUM Always done under radiological supervision. Avoided in bleeding disorders. Risks: hemoptysis, localized bleeding, minor pneumothorax.

56. Lesions in thoracic inlet, hilum and middle mediastinum. Approaches: Supraclavicular Suprasternal Parasternal - Continuous monitoring not possible without real time exposure. - Approaches: Percutaneous route Bronchoscopy Transesophageal endoscopy Spatial orientation Real time monitoring Less risk of radiation exposure.

57. For lesions in subbronchial region Done using fibreoptic bronchoscope Surgical procedures such as mediastinoscopy and surgical biopsy can be avoided. Sensitivity: 56% Specificity: 74% Done using esophageal endoscopy Ultrasound guidance improves diagnostic accuracy Useful in sampling mediastinal lymph node. Rapid technique for diagnosis of pulmonary mass lesions Done under USG or CT guidance 22 G needle is used. Sensitivity: 89% Specificity: 96%

58. ABDOMINAL ORGANS

59. LIVER AND SPLEEN Entry site selected under CT or USG guidance. Based on shortest distance between the skin and the target lesion. Costophrenic angle and major vessels should be avoided.

60. PANCREAS To identify space occupying solid or cystic lesion of pancreas. FNA provides more accurate diagnosis over intraoperative core needle or wedge biopsy of pancreas. Principal approaches: Percutaneous transabdominal aspiration under US or CT guidance EUS guided FNA Direct visualization at the time of laparotomy

61. KIDNEY For definitive diagnosis To reduce surgical exploration Can clarify nature of lesions with equivocal radiological findings Patient is in prone or decubitus position. Large caliber needle is used with obturator as a guide.

62. ADRENAL Patient made to lie prone . Done under USG guidance 20-22 G needle is used. Complications: Surgical emphysema and shock FNAC of pheochromocytoma can lead to fatal hypertensive crisis

63. TRANSRECTAL FNAC OF PROSTATE FRANZEN’S GUIDE AND NEEDLE POSITION OF THE PATIENT

64. TESTIS The patient is placed in the supine position . 1 % xylocaine is injected into the spermatic cord on both sides. 23-gauge needle is used attached to syringe. The needle is inserted into the middle of the anterior surface of the testes opposite the epididymis. The needle is directed in multiple directions to ensure different areas are aspirated.

65. OVARY Usually not done for frank operable ovarian masses. Indicated in: Confirmation of recurrence of malignant tumor To diagnose advanced unresectable ovarian tumor Confirmation of benign incidental ovarian tumor Percutaneous approach Under USG or CT guidance 22 G needle used Proper sampling from representative area sometimes difficult.

66. SKIN, SOFT TISSUE AND BONE

67. SKIN AND SUBCUTANEOUS NODULES Small caliber needle is used. Both FNAC with aspiration and fine needle sampling can be done.

68. SOFT TISSUE LESIONS 22 G needle used Multiple areas sampled Age, duration and size should be considered. Deeper tissue or lesions with bone infiltration should be aspirated under CT or MRI guidance.

69. BONE Not used for primary diagnosis of benign tumors. Yield is usually low especially for cystic lesions. Can be done for malignant bone lesions . Candidates for FNAC chosen after clinical and radiological examination. 22-23 G needles are used. Atleast two passes are done.

70. RECENT ADVANCES IN FNAC Tumor environment mapping(TME) Cellular multiplexing technologies FAST-FNA (fast analytical screening technique fine needle aspiration ) Several molecular tests including in-situ hybridization, polymerase chain reaction (PCR), Southern blotting and gene microarrays. Immuno -FNA grams can rapidly convey the TME landscape and its changes during treatment . A utomated image cytometers: Bioengineering and artificial intelligence (AI) for a rapid analysis. 

71. REFERENCES Orell SR, Sterrett GF . Orell and Sterrett’s Fine Needle Aspiration Cytology. 4th ed. Edinburgh: Churchill Livingstone; 2005 . Gray W, Kocjan G. Diagnostic Cytopathology. 3rd ed. Edinburgh: Churchill Livingstone; 2010 . Dey P. Diagnostic Cytology. New Delhi: Jaypee Brothers Medical Publishers; 2014 . Koss LG, Melamed MR. Koss ' Diagnostic Cytology and its Histopathologic Bases. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2005 . Rane SR, Gadage VS, Bhatia VO, Shinde A. A study of testicular fine needle aspiration cytology (FNAC) in male infertility. Clin Cytol . 2018 Apr 11;4(4 ) Bandyopadhyay A, Chakraborty J, Chowdhury AR, Bhattacharya A, Bhattachrya P, Chowdhury M. Fine needle aspiration cytology of ovarian tumors with histological correlation. J Cytol . 2012;29(1):35-40. doi:10.4103/0970-9371.93218 . Nnodu OE, Giwa SO, Eyesan SU, Abdulkareem FB. Fine needle aspiration cytology of bone tumours --the experience from the National Orthopaedic and Lagos University Teaching Hospitals, Lagos, Nigeria. Cytojournal . 2006 Jun 15;3:16. doi : 10.1186/1742-6413-3-16. PMID: 16776844; PMCID: PMC1526751.