Seminar 7 Cytology 10th Jan 2011

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Cytology

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DIAGNOSTIC CYTOLOGY:

DIAGNOSTIC CYTOLOGY

CONTENTS:

CONTENTS HISTORY & BASICS DEFINITIONS INDICATIONS LIMITATIONS TECHNIQUE ORAL BRUSH BIOPSY FIXATIVE INTERPRETATION COMMONLY USED CYTOLOGICAL STAIN USES OF ORAL CYTOLOGY SMEAR RECENT ADVANCES

History & BASICS :

History & BASICS

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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

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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)

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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 aspiration

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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 mesodermal

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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.”

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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 neoplasia

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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 Cancer

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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 cases

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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.

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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 biopsy

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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)

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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 established

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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 diagnosis

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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 vigorously

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Aspiration

Preparation of smears from aspirates:

Preparation of smears from aspirates Squash prep method Needle spread method Blood smear method

Squash 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 imprint

Common 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.

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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 biopsy

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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 Method

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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 fix

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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.

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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 / PAP

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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 stain

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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 brown

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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 orange

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Interpretation

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A cytologic smear is categorized as falling into one of the five groups Class I : Normal Indicates only normal cells were observed

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PAP smear showing normal Intermediate & superficial Sq.cells

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PAP smear showing normal Intermediate & superficial Sq.cells

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Class II : Atypical Indicates presence of minor atypia but no evidence of malignant changes

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Nuclear enlargements & Variation in size & shape of Nuclei

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Class III : Intermediate This is b/w cancer and non-cancer Cells display wider atypia ; suggests severe dysplasia, carcinoma in-situ BIOPSY IS RECOMMENDED

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Class IV : Suggestive of cancer Few cells with malignant changes; few- many cells may show borderline -characteristics BIOPSY IS MANDATORY

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Pleomorphism ; Irregular N & C outline

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Class V : Positive for cancer Cells show characteristic malignant features BIOPSY IS MANDATORY

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Altered N : C ratio;

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Nuclear pleomorphism

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Cluster of nuclei with little or no cytoplasm

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Squamous epithelial pearl

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Well differentiated Sq.cell ca. Tadpole shaped cells

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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 stains

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Normal basal cells; uniformity in size & shape

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Normal acini of parotid-FNAC

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Smear showing keratin flakes suggestive of a keratocyst

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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 smear

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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 cancer

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Lesions of Pemphigus

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hypertrophic, mononucleated Single/Cluster of supra-basal cells with prominent nucleoli Infl.cells also seen

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Herpes simplex viral infection -- Large numbers of multinucleated epithelial cells Some containing prominent intra-nuclear inclusions

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Herpes simplex viral infection -- Large numbers of multinucleated epithelial cells Some containing prominent intra-nuclear inclusions

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Microbial diseases Fungal – Candida, Actinomyces etc .. colonies can be seen Entamoeba gingivalis parasites – in buccal smears of pts. – poor oral hygiene or following irradiation

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Candidal hyphae

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Anaemia (pernicious, sickle cell)

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Giant/large cells with increased cytoplasm and enlarged central nucleus ; coarsely granular chromatin Bi/multi nucleation can occur

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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 & chemotherapy

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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

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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

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Candida Albicans –Yeast Infection

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Herpes

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Human Papillomavirus - HPV

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Normal Cell Cancerous Cell

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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 cells

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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.

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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 second

REFRENCES:

REFRENCES TEXTBOOK OF ORAL PATHOLOGY- 6 TH EDITION, SHAFER FINE NEEDLE ASPIRATION CYTOLOGY, 4 TH EDITION, SVANTE R ORELL, GREGERY F . STEVETT, DARREL WHITKER

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THANK YOU……

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