PAP SMEARDefinition:- © The BabeS-Papanicolaou test (also called Pap smear, Pap test, cervical smear, or smear test) is a screening test used to detect potentially pre-cancerous and cancerous processes in the the endocervical canal (transformation zone) of the female reproductive system. © Credit for its development goes to Dr George N. Papanicolaou. dt. monika nemaPatient preparation ® Women should be tested two weeks after the first day of their last menstrual period.(Day |4 of cycle is optimal). ® Women should not use any vaginal medication, contraceptive during the 48 hrs prior to sample collection. ® Sexual relationship is not recommended the night before the test.Specimen collection ® Specimens should be obtained after a nonlubricated speculum (moistened only with warm water if needed) is inserted * Excess mucus or other discharge should be removed gently with ring forceps holding a folded gauze pad. * An optimal sample includes cells from the ectocervix and endocervix(| Squamo-Columnar Junction ( el * Junction of pink cervical skin and red endocervical canal ® Inherently unstable ® Key portion of the cervix to sample ® Most likely site of dysplasia® The location of the squamo-columnar junction (8 mm to 13 mm proximal to the external cervical OS) in most women varies with the age and fertility. dr. monika nemaCOLLECTION OF SAMPLE eee ear Petey PB Pc) ® eee ty er ay = ori Gr cdAyers Spatula ® Concave end to fit the fa cervix ® Convex end for vaginal wall and vaginal pool scrapings. ® One end is longer so that spatula fits the external OS.Metaplastic cells Squamous cells i ae a we Columnar cells Figure 5.3. Endocervical sampling by means of an Ayre-type instrument requires a 360° rotation of the instrument under pressure. The principal cell types secured by the instrument are also shown. dr. monika nema® Easier to introduce into narrow cervical canal because the hairs fold down along the shaft and is likely to retain cells. ® Insert ~ 2 cm (until brush is fully inside canal) ® Rotate only 180 degrees (otherwise will cause bleeding)‘f & —eridocenvical eptheliuan Ve a Ayre's spatula Figure 5.1, Methods of securing cytologic samples from the {female genital tract. a Vaginal smear from the posterior cul-de- ‘ac, the original Popanicolaou method. Besides cells rom the vagina and cervix, the samples may contain endometial cells and cells from the unper reaches ofthe genital trac. b Smear from the lateral wall ofthe vagina for hormonal assessment. -cAyre’s spatula fr cervical smears. dA medication of Ayre's spatula with a longer endocervical arm to improve endocervi- ‘cal sampling, e Endocervical brush instrument, ds, monika nemaMake Pap Smear ® As thin as possible ® Properly labeledFixation © Fixative is an agent used to prepare cytologic specimen for the purpose of preserving and maintaining the existing form and structure of all constituent elements.© 95% Ethanol. © 95% Rectified Spirit. © 100% Methanol. ® 80% Isopropanol or Propanol. ® Ether/95% Ethanol ( |: 1). ® Spray fixatives contains isopropanol and propylene glycol.Figure 5.4. Spray fixation of smears. The optimal distance between the nozzle of the spray can and the slide is approxi- mately 10 inches (30 cm). immediate fixation (within seconds) is critical in order to prevent air-drying artifactPAP stain © 80% alcohol — 6 to 8 dips. © 70% alcohol — 6 to 8 dips. © 50% alcohol — 6 to 8 dips. ® Distilled water - 6 to 8 dips. © Harris Haematoxylene — 6 minutes. © 0.25% HCI- 6 to 8 dips. © Running tap water — 6 minutes. ® 50% alcohol — 6 to 8 dips. © 70% alcohol — 6 to 8 dips. © 80% alcohol — 6 to 8 dips. ® 95% alcohol — 6 to 8 dips. © O.G.G- 2 minutes. dr, monika nemaPAP stain © 95% alcohol — Rinse in two dishes. © E.A5O- 2 minutes. © 95% alcohol — Rinse in three dishes. ® 100% alcohol - 6 to 8 dips. © 100% alcohol — 6 to 8 dips. © Equal parts of absolute alcohol and xylene— Rinse in two dishes. © Xylene- 6 to 8 dips © Xylene—6 to 8 dips. © Xylene— 6 to 8 dips © DPX mount dr, monika nema( Liquid based cytology ® The liquid based cytology technique involves rinsing all the material collected on the sampling device into a fixative fluid, creating a cell suspension. | ® Liquid sample is sent to the laboratory rather than glass slide preparation.Liquid-based monolayer cytology ® Two of the types -Sure-Path (TriPath Imaging) -Thin-Prep (Cytyc Corp). © The media are pee ee peat bated for Sure- Path and methanol for ThinPrep.fe LIQUID BASED CYTOLOGY Different types of brushes allowing to collect cells fi ‘om the ectocervix and endocervix. ke Ectocervix and 0.5 om of endocervix are sampled The central bristles of the broom are inserted into the endocervical canal until the lateral bristles bend fully against the ectocervix The sampling device is rotated 360 degree in the clockwise direction twice and then anticlockwise while maintaining gentle pressure,The brush is removed and deposited in the methanol based liquid medium.RBC are lysed by transport medium.L.DISPERSION 2, CELL COLLECTION 3. CELL TRANSFER The entire procedure takes about 70 seconds per slide and results in a thin deposit of cells in a circle 20 mm in diameter.Sure path ® Samples are collected in ethanol based transport medium. ® The tip of the collection device is snipped off and included in the sample vial. ® The equipment to prepare slides includes a Hettich centrifuge and a PrepStain robotic sample processer with computer and monitor.Red blood cells and some leukocytes are eliminated by density centrifugation. * In addition to preparing an evenly distributed deposit of cells in a circle 13 mm in diameter, the method incorporates a final staining step that discretely stains each individual slide.LIQUID BASED CYTOLOGY * Reduces number of inadequate smears and need for repeat smears * Thin-Prep appears to be superior to convention Pap test in detecting SIL. * Cellular preservation is enhanced. * Good fixation results in improved microscopic details.with the newka, 2 ; Ses microscope: Notice the cloth bi ack galt gund and how well the cells are SS = ee lt a’ iw oF ler dl 3 a e 10xCervical cytology practice guidelines Test Requisition he minimal clinical data Age Date of LMP or onset of menopause Past or Current history of any abnormalities or treatment Pregnancy status ir. monika nemaFigure 5.5. Three methods of screening smears. In the authors’ opinion, method 2 is the best because it allows screening of overlapping fields. Method 3 is recommended by some observers for rapid rescreening as a quality control measure. dr, monika nemaNORMAL CaicaL Ces ite cells my f Basal cells ets oi. .* OPE be 3 < a eeParabasal Cells ® Small round cells with round nuclei and small amount of cytoplasm ® Uniform in size and shape dr. monika nemaIntermediate Cells ® May be small or large ® Round nuclei, nucleus similar in size as parabasal cells ® Entire cell approximately twice the size of parabasal cells ® Cytoplasm becomes angular, irregular and folded as cell enlargesSuperficial Cells © Largest epithelial cell ® As they age and degenerate, the nuclei becomes small, pyknotic and fades. ® Cytoplasm may contain vacuoles with ageSuperficial Cells Continued ® Cornification is the degeneration process ® Superficial cells are commonly called cornified cells ® Once nucleus is lost become Anuclear cellsBethesda System 2001 © Specimen type ® Indicate conventional smear (Pap smear), © liquid based preparation or other preparation (describe) © (A) Statement of adequacy of the specimen. ® Satisfactory © Satisfactory for evaluation but limited by © Unsatisfactory. © (8) General categorization of the diagnosis . © Within normal limits. © Benign cellular changes. © Epithelial cell abnormality. ® (C) Descriptive diagnosis © Infections © Reactive changes ® Epithelial cell abnormalities © Other malignant neoplasms ® Hormonal evaluation. dt. monika nemaBethesda System 2001 © Interpretation/result © Negative for intraepithelial Lesion or Malignancy (NILM) © Organisms © © Trichomonas vaginalis © Candida species © = Bacterial vaginosis ‘© © Actinomyces species © + Herpes simplex virus (Other non-neoplastic findings- Reactive cellular changes associated with © = inflammation (includes typical repair) © irradiation © = Intraurerine contraceptive device (IUD) dt. monika nemaBethesda System 2001 © Epithelial Cell Abnormalities ® SQUAMOUS CELL © © Atypical squamous cells + of undetermined significance (ASC-US) = cannot exclude HSIL (ASC-H) “Low grade squamous intraepithelial lesion (LSIL) encompassing HPV/mild dysplasia/CIN | ‘High grade squamous intraepithelial lesion (HSIL) encompassing: moderate and severe dysplasia/CIN2/CIN3/CIS = With features suspicious fer invasion (if invasion suspected) * Squamous cell carcinoma © GLANDULAR CELL Atypical glandutar cells of uncertain significance ‘Atypical glandular cells- endocervical, endometrial NOS AGC, favor neoplastic ® + Endocervical Adenocarcinoma in situ © © Adenocarcinoma endocervical + endometrial - extrauterine dt. monika nemaStatement of Specimen Adequacy dt, monika nema| _ Adequate smear ® An adequate pap smear is one that includes a sampling of both the exocervix and endocervix. ® An adequate cytologic sample contains more than 300 squamous cells, including at least two clusters of 5 cells each of endocervical and/ or metaplastic cells with mucus material.This image was composed to depict the appearance of a 4X field with approximately 1400 cells. It is to be used as a guide in assessing squamous cellularity of conventional specimens. An adequate conventional smear has an estimated minimum of approximately 8,000-12,000 well visualized and preserved squamous cells,Cytomorphologic Criteria: Satisfactory squamous cellularity on a ThinPrep slide. Endocervical cells are also present. An adequate liquid based preparation should have an estimated minimum of 5,000 well- visualized/preserved squamous cells. de. monika nemaCytomorphologic Criteria: Distinct cytoplasmic borders are seen in the cluster of cells on the left, giving a ?honeycomb? appearance. The cell cluster on the right is seen. from a side view, giving the ?picket fence? appearance. Interpretation is NILM. ir, monika nemaThis specimen is unsatisfactory due to scant squamous cellularity seen attOX de. monika nemaCytomorphologic Criteria: Over 75% of cells are obscured by inflammation and blood. dr. monika nemaDefination and criteria for specimen adequacy“Satisfacotry for evaluation” © Approriate labelling and identifying information. ® Relevant clinical information. ® Adequate numbers of well preserved and well visualized squamous epithelial cells. ® An adequate endocervical transformation zone component.Satisfactory for evaluation but limited by.... ® Lack of minimum clinical patient information. © Partially obscuring blood, inflammation,thick areas,poor fixation etc that precludes interpretation of approximately 50% to 75% of the epithelial cells. ® Lack of endocervical / transformation zone component.Unsatisfactory for evaluation... ® Lack of patient identification on specimen. ® Slide that is broken and cannot be repaired, or cellular material that is inadequately preserved. ® Scant squamous epithelial component (well preserved and well visualized squamous epithelial cells covering less than 10% of the slide surface) Obscuring that precludes interpretation of approximately 75% or more of epithelial cells.® Any epithelial abnormality is of paramount importance and must be reported regardless of compromised specimen adequacy. ® lf abnormal cells are detected, the specimen is never categorised as “ UNSATISFACTORY”Infections dr, monika nemaLACTOBACILLI population. These bacilli release containing cells: . Mainly affect intermediate and superficial cells. Parabasal cells are generally spared. Gr, monika nemaBacterial vaginosis © Between puberty and the menopause the presence of lactobacilli maintains a pH of vagina between 3.8 and 4.2. ® Changes in pH over 4.5 leads to non specific vaginitis. ® It is an infectious disease classically associated with gray or white,thin, homogenous discharge that tends to adhere to vaginal walls. © Exudes a characteristic fishy odour when mixed with 10% KOH. © Gardnerella vaginalis — putative pathogen.{ Bacterial vaginosisiie, Interpretation: NILM: Shift in Flora suggestive of bacterial vaginosis ‘dr. monika nemaHerpes simplex Cytomorphologic Criteria: Nuclei showing "ground-glass" appearance. Multinucleation, Huclear molding, and dense eosinophilic intranuclear inclusions surrounded by a halo are also seen.. © Cytomorphologic Criteria: Tangled clumps of filamentous organisms, often with acute angle branching, sometimes showing irregular wooly appearance. Swollen filaments may be seen with clubs at periphery. A cotton ball like acute inflammatory response is common. Actinomyces is often associated with intrauterine device (IUD) usage. dt. monika nemaCytomorphologic Criteria: Pseudohyphae and reactive changes in the squamous epithelial cells. ds. monika nemaTrichomonas vaginalis s oe Cytomorphologic Criteria: Trichomonas is a pear-shaped, oval to round, cyanophilic organism that ranges in size from 15-30 microns. The nucleus is pale, vesicular and centrally located. Eosinophilic granules are often visible in the cytoplasm. dt. monika nemaAcute inflammation © Dirty background of the smear. ® Desquamation of cells in sheets and aggregates. © Large number of degenerating polymorphs. ® Dark pyknotic nuclei in superficial and intermediate cells. © Perinuclear halos. ® Increased number of parabasal cells. © Enlarged endocervical cells with prominent chromocentres.INFLAMMATORY LESIONS ACUTE INFLAMMATIONCytomorphologic Criteria: Anucleate mature polygonal squamous cells with ghost-like ?nuclear holes? ‘de. monika nemaCytomorphologic Criteria: Polymorphous population of lymphoid cells and tingible body macrophages. dr. monika nema‘REACTIVE CHANGES IN SQUAMOUS “CELLS IN INFLAMMATORY LESIONS oe or * sr, * wee: +, i Hor & a = »- Phd ~*. ‘. & i”. — A am 2 ce hy weer. ies ‘se ? he * say 3 nahi ey “ e 3 ~ at eeLAMMATORY CHANGES ININFLAMMATORY CHANGES IN ENDOCERVICAL CELLS tl Cytoplasmic a a Dg endocervical cells 2 feIUD EFFECT »o ¢ Smail cluster of glandular cells with cytoplasmic vacuoles displacing nuclei,creating a signet-ring appearanceir mmoniika nema Overall increase in cell size Cytoplasmic vacuolation, Uneven staining of cytoplasm. Nuclear enlargement with vacuolation. Multinucleation, Fragmentation of nuclei.Endometrial cells\ | Epithelial cell abnormality ® The diagnosis of ASCUS ( Atypical squamous epithelial cells of undetermined significane) is offered only when the cytomorphological changes exceed the parameters related to benign,reactive processes but fall short of a definite diagnosis of a squamous intraepithelial lesion.ASC-US ® Cells resemble superficial or intermediate cells. ® Nuclear size is increased 2-3 times. © Nuclear boundraies are regularor with minimum irregularities. dr. monika nemaASC-H (cannot exclude high grade intraepithelial lesion) ® Cells resemble parabasal or basal in configuration and size. ® Nuclei are hyperchromatic with uneven chromatin pattern. ® Nuclear membrane is thick and uneven.ASCUS- MATURE~ ASC-H (cannot exclude high grade intraepithelial lesion) ~~ - » 'é | cosely cahes|ve metanlasils-calls with ET-H mula eon cells and slightly enlarged nuclei with occasional nugi@ar contour irregularities. oo f 5 ow . dr. monika nemaLow-grade squamous High-grade squamous intraepithelial lesion (LSIL) intraepithelial lesion (HSIL) : Moderne ere | In Situ Normal Very mild to mild dysplasia a tte ee eeeSO COMPARISON OF CLASSIFICATIONS CIN WHO GRADE | Mild dysplasia ll Moderate dysplasia ll Severe dysplasia BSCC BETHESDA borderline ASCUS Mild Low grade dyskaryosis SIL Moderate —_ High grade dyskaryosis SIL Severe High grade dyskaryosis SIL@ In CIN | and Ill, the characteristic nuclear changes are better observed in intermediate cells. ® As the grades of CIN increase, parabasal and basal cells start showing nuclear abnormalities. ds. monika nemaCervical intraepithelial neoplasia -| % oP . Slight nuclear enlargement Hyperchromasia but finely granular chromatin. nuclear membrane is smooth and without indentation. dr. monika nemaCervical intraepithelial neoplasia -lll te ae alle 4 Hyperchromasia is more marked. Chromatin can be seen in small clumps. Nuclear membrane is thickened slightly but indentation are ususlly absent.Cervical intraepithelial neoplasia lll Nuclear membrane is characteristically irregular and indented dr. monika nemaHPV infection ® Seen in most of the cases of CIN. ® Lesions of CIN | and CIN Il are usually positive for HPV 6,1 1,31,42. ® Lesions of CIN III and invasive cancer are usually positive for HPV 16,18,33.ILOCYTE EHP)DOKOILOCYTES cogen in squamous ee a {fy dokoilocytosis". lear abnormalities ired for an pretation of ASC-US/ are absent. ir mmonilga nersSquamous cell carcinoma- non keratinizing , regular chromatin, rominent nucleolus Dysplastic squamous cells with anisocyt a including keratinization and tadpole a of invasive squamous cell carcinoma.Squamous cell carcinom- keratinizing Tadpole cell / yfpical endocervical cells 3 so round or oval nucle with prominent Mitotic figures Sheet of cells with enlarged, variably-sized nuclei with some crowding and overlap of nuclei.ENDOCERVICAL ADENOCARCINOMA IN SITU (AIS) Atypical columnar endocervical cells, with enlarged, elongated and hyperchromatic nuclei » ical feathering and palisading. Hing Explanatory Notes: Pseudostratification, nuclear crowding and feathering are classic features of AISADENOCARCINOMA » Cytomorphologic Criteria: Cluster of cells with enlarged round or oval nuclei, irregular chromatin distribution and prominent nucleoli. Explanatory Notes: Irregular chromatin distribution and prominent or macronucleoli are a classic findings in invasive endocervical adenocarcinoma. dt. monika nemaINVASIVE ADENOCARCINOMA; Re Cells are round, crowdecINVASIVE ADENOCARCINOMA Crowding, large nucleo ZwENDOMETRIAL ADENOCARCINOMA Large hyperchromatic nucleus, Vacyolated cytoplasmHormonal cytology ® Maturation of vaginal squamous cells form one cell to another is hormone dependent. ® The quantitative ratio between the different cell types can reflect the index of the hormonal status of the female. ® For hormonal assesment ideal site is lateral vaginal wall.© Estrogens have proven action on Wa maturation of squamous epithelium of vagina. © Its excess causes enhancement of maturation and the smear contains more of superficial cells, on the other hand its lack causes lower degree of maturation or the atrophy of squamous epithelium, the same effect could be reflected due to antagonistic action of the excess of progesterone.Cellular indices for hormonal assessment ® Karyopyknotic index ® Eosinophilic index ® Folded cell index ® Crowded cell index ® Maturation indexKaryopyknotic index \ ® Ratio between the superficial squamous cells with pyknotic nuclei to all mature squamous cells irrespective of staining character ® Peak of KPI usually coincides with the time of ovulation and may reach 50-85.| _ Eosinophilic index ® Ratio of mature squamous cells with eosinophilic cytoplasm to all mature squamous cells irrespective of size of nucleus. ® Peak value is 50-75 during ovulation.Folded cell index ® Ratio of mature squamous cells with folded margins to all mature squamous cells irrespective of staining chararcter. ® Folding is usually observed in cells containing glycogen.Crowded cell index © Represents the relationship of mature squamous cells lying in clusters of four or more cells, to all mature squamous cells. dr. monika nemaMaturation index ® It is count of the parabasal cells, intermediate and superficial cells . @(P:1:S) ® In a normal menstruating woman during ovulation the menstruation index will be 0/35/65. © In postmenopausal it will be 85/15/0.Maturation value ® Meisels suggested that a value be given to each category of cells i.e, value of 1.0 to superficial cells, 0.5 to intermediate cells and 0.0 to parabasal cells. ® This system gives single value ranging from 0-100 to express hormonal status.© Maturation value 100 — purely superficial cells. 0 — purely parabasal cells. 50-95 — in normal menstruating woman. < 50 — atrophic squamous epithelium. dr. monika nemaDIFFRERENT PHASES OF MENSTRUAL CYCLE ESTROGEN PHASE ~~ . . +, ie . . 2S ge - e 2 eosihophitic Supereal squamous, J cellssclean, 4 2 pe abot ae . Bal. a E Ve * . ; 5 . * . * 5 és oh “a é iment a a - . Sere aS SS eee 5 ee” “cg E a te , alOVULATORY SMEAR we oe a pore } £ a a ‘ r J roe: 44 in My fs = * forn-tike crystalline structures a = : ms &. eo |! : es ? & - \) Cosinophilic superficial a, = s (squamous cells ne a ve bile A ap: ada Peo ~ basophilic squamous cells with folded cytoplasm (boat cells) > Glycogen depositsPROGESTERONE PHASE WITH ABUNDANT LACTOBACILLI AND CYTOLYSISMenopause ® Maturation of cervical epithelium is estrogen dependent ® Due to its insufficiency, maturation is retarded. ® Number of mature squamous and intermediate cells is reduced. © In early menopause intermediate cells increase in number.Cytomorphologic Criteria: Parabasal cells and blue blobs. Explanatory Notes: Overall cellularity of smear is reduced. Small and large parabasal cells are seen with almost absence of superficial cells. Blue blobs are globular collections of basophilic amorphous material reflecting either degenerated parabasal cells or inspissated mucus dt. monika nemaAmercian cancer society recommendations ® Screening should begin ne later than age 21. © Screening should begin earlier than age 21 if the patient is sexually active. In this case, it should start 3 years after initiation of vaginal intercourse. Once initiated, screening should be performed annually. After age 30, for women who have had 3 consecutive normal pap smears, screening frequency may be reduced to every two to three years. Women who are HIV positive, inmunocompromised should continue annual screening. Patient tested positive for HPV, should continue to be screened indefinitely. ® May stop after age 70, if patient is low risk and has had three normal pap smears over the last 10 years.® In developing countries where there are no organized screening programme for pap test due to financial constraints, poor infrastructure or inadequate human resources, the WHO recommends five yearly five pap tests during life span of 35 to 55 years.THANK YOU SPEAKER : Dr. MONIKA NEMA dr, monika nema