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Premium member Presentation Transcript DIAGNOSTIC CYTOLOGY: DIAGNOSTIC CYTOLOGYCONTENTS: CONTENTS HISTORY & BASICS DEFINITIONS INDICATIONS LIMITATIONS TECHNIQUE ORAL BRUSH BIOPSY FIXATIVE INTERPRETATION COMMONLY USED CYTOLOGICAL STAIN USES OF ORAL CYTOLOGY SMEAR RECENT ADVANCESHistory & BASICS : History & BASICS: Historical background Origins of Cytopathology – century old European microscopists – observed – morphologic differences b/w human cells in health & disease subsequent studies demo. That cancer could be diagnosed by study of cells shed in body fluids/ obtained by aspiration: Until Land mark report by Dr. George N. Papanicolaou (1943) on detection of carcinoma of uterine cervix in vaginal smears But not gained importance (b’se most believed in tissue diagnosis): Definitions Cytology :- “ Cytology is the art & science of interpretation of cells ” that either exfoliate Freely / naturally from the surface or obtained from the tissue by various clinical procedures such as Scraping/brushing, washings or by fine needle aspirationPowerPoint Presentation: Cytopathology :- “Cytopathology is the study of the structural changes in the disease-altered desquamated or aspirated cells from various sites.” The majority of cells obtained are epithelial; few mesodermalPowerPoint Presentation: Oral Exfoliative Cytology “Art & science of interpretation of cells specifically from oral cavity which have either been exfoliated or obtained by scraping from mucosa or by fine needle aspiration.”PowerPoint Presentation: RATIONALE : Normal epithelial cells – physiologic turnover EC is based upon the rapid / abnormal proliferation of malignant epithelial cells – shed quickly / easily due to lack of cohesiveness. Thus, cells obtained – reliable indicators of dysplasia or neoplasiaPowerPoint Presentation: A Mechanism for Oral Cancer Development Damage to DNA HPV Environmental Carcinogens Tobacco Carcinogens Alcohol Abuse DNA Repair Cell Growth Regulation DNA Content Apoptosis Nuclear Instability Oral CancerPowerPoint Presentation: Indications To diagnose a suspicious area in those pt’s not referred for biopsy Large, multiple, varied lesions Early detection of cancer Rapid diagnosis – Screening Follow up -- detection of recurrences -- treated casesPowerPoint Presentation: Indications To assure the overly apprehensive pt. Forensic dentistry – Sex determination (Barr bodies in females) Infections viral - Viral inclusion bodies , fungal – fungal hyphae can be seen Vesicular lesions –Pemphigus , HSV.PowerPoint Presentation: Cytopathology Vs Histopathology Advantages Provides a rapid, inexpensive, & simple means of diagnosis Little tissue injury - frequent sampling - evaluation of progression to R x / recurrences Better accepted by the patient & physician Samples cells from wider surface than a biopsyPowerPoint Presentation: Cells can be obtained from inaccessible/ difficult to access areas Minimum shrinkage or distortion of cells Determination of hormonal states Smears permit better evaluation of the nature of inflammations and infections (microorganisms are easily seen)PowerPoint Presentation: Limitations Cytologic diagnosis not always final; must often be confirmed by histology Diagnosis is based upon the study of minute cellular details – tissue patterns cannot be appreciated – Interrelation and arrangement of the cells to the supporting stroma cannot be establishedPowerPoint Presentation: Location of lesion cannot be pin-pointed (except in FNAC) Size of the lesion cannot be estimated Error / misinterpretation may occur Limitations Lack of cellular architecture Inadequate sample/cell yield Expertise interpretation May need confirmatory biopsy in malignancy Limits the diagnosisPowerPoint Presentation: Technique Relatively simple one. Cleanse the area for debris / mucin etc., Using a metal cement spatula /tongue blade – scrape the entire surface of the lesion several times vigorouslyPowerPoint Presentation: AspirationPreparation of smears from aspirates: Preparation of smears from aspirates Squash prep method Needle spread method Blood smear methodSquash Preparation: Squash Preparation With experience, can yield excellent cytologic smears Aspirated material is placed on the center of the slide A second slide is placed over the sample to form a cross. Carefully slide apart from first slide (Put down on and pick up to move). Do not place excessive downward pressure to the first slide because will cause distorted ruptured cells The weight of the spreader slide is sufficient to adequately spread the cells.Needle Spread Method: Needle Spread Method Spread aspirate on the slide with tip of needle. Pull sample out into several projections (starfish appearance).Blood Smear Technique: Blood Smear Technique Use if material is thick or fluid After material is expelled on slide, second slide is held at 30-40˚angle. Second slide is pulled backward until it contacts the fluid Rapidly move forward like a blood smear.Common Problems with FNA: Common Problems with FNA Few or no cells obtained Some lesions do not exfoliate cells well. The needle may miss the site of the lesion Timid collection Inadequate negative pressure Blood contamination Using too large needle gauge Prolonged aspiration Failure to blot if doing imprintCommon Problems with Preparation: Common Problems with Preparation Poorly prepared slides due to thick or high cell numbers Allowing material to dry on slide before squash prep or other smear technique. If a large amount of material is present, spread between two slides May have to do 4-5 slides form the same site in order to get valuable diagnostic sample.PowerPoint Presentation: Special instrument called biopsy brush Trans-epithelial biopsy obtained Indications For precancerous / cancerous oral mucosal lesions Advantages Easy to perform; requires less time Well tolerated by the patient Oral Brush biopsyPowerPoint Presentation: Oral Cells From Brush Phosphate Buffered Saline pH 7.4 Flow Cytometric Analysis DNA Content-”Ploidy” 2. Cell Cycle,Apoptosis, etc. Novel Extension of Current MethodPowerPoint Presentation: A wet fixed smear gives better presentation of nuclear details (routinely preffered) A smear is air dried only when specifically indicated (Mc. Grunwald-Giemsa stain (MGG), immunocytochemical procedures) Prepare a smear (Quickly spread the collected – evenly over a microscopic slide & ) Fix immediately before the smear dries – wet fixPowerPoint Presentation: Fixative s: Fluid fixatives. 95% alcohol Equal parts of alcohol & ether Spray fixative. Contain water-soluble polymer/plastic Most contain- polyethylene glycol. Hair spray with high alcohol can be used.PowerPoint Presentation: After flooding with fixative, allow to stand for 30min, air dry, stain Always minimum of 2 slides are prepared (scraped separately) Staining : For routine diagnostic cytology , the universal stain is Papanicolaou stain / PAPPowerPoint Presentation: PAP smear: Dr. George N. Papanicolau – Credited with introduction of cytologic method for diagnosis of Carcinoma of Cervix – 1942. Modified further in – 1954 & 1960 Smear and stain both – named after him. - PAP smear - PAP stainPowerPoint Presentation: Used universally throughout cytology. Rationale / Basis: Differentiation and maturation of cells – show characteristic morphologic features, which can be identified by staining. PAPANICOLAOU STAIN A multichromatic stain uses aqueous hematoxylin with multiple counterstaining dyes – Eosin, Orange G, Light green, Bismarck brownPowerPoint Presentation: Results of PAP stain: Nuclei –shows Health of cell – Appears blue – Hematoxylin Cytoplasm – Shows functional differentiation and origin. As it proceeds from basal surface layer, Color transition from – Blue / Green Pale orange / pink Dark orangePowerPoint Presentation: InterpretationPowerPoint Presentation: A cytologic smear is categorized as falling into one of the five groups Class I : Normal Indicates only normal cells were observedPowerPoint Presentation: PAP smear showing normal Intermediate & superficial Sq.cellsPowerPoint Presentation: PAP smear showing normal Intermediate & superficial Sq.cellsPowerPoint Presentation: Class II : Atypical Indicates presence of minor atypia but no evidence of malignant changesPowerPoint Presentation: Nuclear enlargements & Variation in size & shape of NucleiPowerPoint Presentation: Class III : Intermediate This is b/w cancer and non-cancer Cells display wider atypia ; suggests severe dysplasia, carcinoma in-situ BIOPSY IS RECOMMENDEDPowerPoint Presentation: Class IV : Suggestive of cancer Few cells with malignant changes; few- many cells may show borderline -characteristics BIOPSY IS MANDATORYPowerPoint Presentation: Pleomorphism ; Irregular N & C outlinePowerPoint Presentation: Class V : Positive for cancer Cells show characteristic malignant features BIOPSY IS MANDATORYPowerPoint Presentation: Altered N : C ratio;PowerPoint Presentation: Nuclear pleomorphismPowerPoint Presentation: Cluster of nuclei with little or no cytoplasmPowerPoint Presentation: Squamous epithelial pearlPowerPoint Presentation: Well differentiated Sq.cell ca. Tadpole shaped cellsPowerPoint Presentation: Routine cytology Cancer cells & precursors – PAP stain Bacteria -- Gram’s, acid fast Viruses -- immunocytochemistry , direct immunofluorescence Fungi -- PAS, Gorcott’s methanamine silver Lipids -- Oil red O Commonly used cytological stainsPowerPoint Presentation: Normal basal cells; uniformity in size & shapePowerPoint Presentation: Normal acini of parotid-FNACPowerPoint Presentation: Smear showing keratin flakes suggestive of a keratocystPowerPoint Presentation: With respect to cancer, Numerous studies on Pt’s with oral cancer Concluded that, Cytology is an adjunct, but not a substitute for the surgical biopsy Quick, simple, painless, helpful in follow-ups; screening Check against false – ve biopsies Uses of oral cytology smearPowerPoint Presentation: which are characterised by presence of certain specific cells vesicular lesions Pemphigus Herpes Infections - Candida Anemia Cell reactions to irradiation, chemotherapy Herditary diseases like benign intra epithelial dyskeratosis , white sponge nevus Also for evaluation or Δ sis of diseases other than cancerPowerPoint Presentation: Lesions of PemphigusPowerPoint Presentation: hypertrophic, mononucleated Single/Cluster of supra-basal cells with prominent nucleoli Infl.cells also seenPowerPoint Presentation: Herpes simplex viral infection -- Large numbers of multinucleated epithelial cells Some containing prominent intra-nuclear inclusionsPowerPoint Presentation: Herpes simplex viral infection -- Large numbers of multinucleated epithelial cells Some containing prominent intra-nuclear inclusionsPowerPoint Presentation: Microbial diseases Fungal – Candida, Actinomyces etc .. colonies can be seen Entamoeba gingivalis parasites – in buccal smears of pts. – poor oral hygiene or following irradiationPowerPoint Presentation: Candidal hyphaePowerPoint Presentation: Anaemia (pernicious, sickle cell)PowerPoint Presentation: Giant/large cells with increased cytoplasm and enlarged central nucleus ; coarsely granular chromatin Bi/multi nucleation can occurPowerPoint Presentation: Cytoplasmic & nuclear degeneration is seen pyknosis , karyorrhexis , foamy vacuolization Regeneration produces marked enlargement of cytoplasm & nuclei; Moderate increase of inflammatory cells & epi.phagocytes usually present Cell reactions to irradiation & chemotherapyPowerPoint Presentation: Cytoplasmic & nuclear degeneration is seen pyknosis , karyorrhexis , foamy vacuolization Regeneration produces marked enlargement of cytoplasm & nuclei; Moderate increase of inflammatory cells & epi.phagocytes usually presentPowerPoint Presentation: Cytoplasmic & nuclear degeneration is seen pyknosis , karyorrhexis , foamy vacuolization Regeneration produces marked enlargement of cytoplasm & nuclei; Moderate increase of inflammatory cells & epi.phagocytes usually presentPowerPoint Presentation: Candida Albicans –Yeast InfectionPowerPoint Presentation: HerpesPowerPoint Presentation: Human Papillomavirus - HPVPowerPoint Presentation: Normal Cell Cancerous CellPowerPoint Presentation: It should remembered that most Benign neoplasms of oral cavity do not lend themselves to cytologic smears Eg :- fibroma – cytology is rarely diagnostic(unless ulcerated) – ve cytology report should be confirmed by a repeat cytology / biopsy in clinically suspicious cases Leukoplakia ( hyperkeratotic lesions) – scarcity of viable surface cellsPowerPoint Presentation: Recent advances Liquid based thin layer slide preparation Cells collected with brush / other instrument rinsed in a vial of liquid preservative. Sent to lab Automated thin layer slide device prepares slide for viewing.PowerPoint Presentation: Recent advances Automation of cytologic screening Use of powerful computers Cyto-analyzer – based on measurements various cellular parameters Image analysis – quantitative analysis of various cell components Flow cytometry – can measure multiple physical characteristics of cells suspended in a solute at a rate of 3000 – 5000 cells per secondREFRENCES: REFRENCES TEXTBOOK OF ORAL PATHOLOGY- 6 TH EDITION, SHAFER FINE NEEDLE ASPIRATION CYTOLOGY, 4 TH EDITION, SVANTE R ORELL, GREGERY F . STEVETT, DARREL WHITKERPowerPoint Presentation: THANK YOU…… You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.