PAP SMEAR

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Definition:-The Babe -Papanicolaou testș  (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.

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Patient preparationPatient preparationWomen should be tested two weeks

after the first day of their last menstrual period.(Day 14 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.

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Specimen collectionSpecimen 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

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Squamo-Columnar JunctionSquamo-Columnar Junction

Junction of pink cervical skin and red endocervical canal

Inherently unstable Key portion of the

cervix to sampleMost 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.

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COLLECTION OF SAMPLECOLLECTION OF SAMPLE

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Ayers SpatulaAyers Spatula

Concave end to fit the cervix

Convex end for vaginal wall and vaginal pool scrapings.

One end is longer so that spatula fits the external OS.

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CytobrushCytobrush

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)

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Make Pap SmearMake Pap SmearAs thin as possibleProperly labeled

Fixation 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.

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95% Ethanol.95% Rectified Spirit.100% Methanol.80% Isopropanol or Propanol.Ether/95% Ethanol ( 1: 1).Spray fixatives contains isopropanol and

propylene glycol.

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Immediate fixation (within seconds) is critical in order to prevent air-drying artifact

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PAP stainPAP 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% HCl- 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.

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PAP stainPAP stain 95% alcohol – Rinse in two dishes. E. A 50 - 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

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Liquid based cytologyLiquid based cytologyThe 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.

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Liquid-based monolayer cytologyLiquid-based monolayer cytologyTwo of the types -Sure-Path (TriPath Imaging) -Thin-Prep (Cytyc Corp).

The media are primarily ethanol-based for Sure-Path and methanol for ThinPrep.

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Different types of brushes allowing to collect cells Different types of brushes allowing to collect cells from the ectocervix and endocervixfrom the ectocervix and endocervix. .

LIQUID BASED CYTOLOGY

Ectocervix and 0.5 cm 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.

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The brush is removed and deposited in the methanol The brush is removed and deposited in the methanol based liquid medium.RBC are lysed by transport based liquid medium.RBC are lysed by transport medium.medium.

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Thin Prep methodThin Prep method

The entire procedure takes about 70 seconds per slide and results in a thin deposit of cells in a circle 20 mm in diameter.

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Sure pathSure pathSamples 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.

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• 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.

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LIQUID BASED CYTOLOGYLIQUID 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.

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Conventional smear

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What we see under the microscope conventional smear

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The prepared slide with the new ThinPrep

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What we see under the microscope. Notice the clean back ground and how well the cells are dispersed rendering easier to interpretation.

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Test Requisition

The minimal clinical data:

Age

Date of LMP or onset of menopause

Past or Current history of any abnormalities or treatment

Pregnancy status

Cervical cytology practice guidelines

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Superficial cells Intermediate cells

Parabasal cells Metaplastic cells

NORMAL CERVICAL CELLSNORMAL CERVICAL CELLS

Basal cells

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Parabasal CellsParabasal CellsSmall round cells with round nuclei and

small amount of cytoplasmUniform in size and shape

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Intermediate CellsIntermediate CellsMay be small or largeRound 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 dr. monika nema

Superficial CellsSuperficial CellsLargest epithelial cellAs they age and degenerate, the nuclei

becomes small, pyknotic and fades.Cytoplasm may contain vacuoles with age

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Superficial Cells ContinuedSuperficial Cells ContinuedCornification is the degeneration processSuperficial cells are commonly called

cornified cellsOnce nucleus is lost become Anuclear

cells

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BethesdaBethesda System 2001System 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. (B) 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.

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BethesdaBethesda System 2001System 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      - Intrauterine contraceptive device (IUD)

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BethesdaBethesda System 2001System 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 I

• High grade squamous intraepithelial lesion (HSIL)

- encompassing: moderate and severe dysplasia/CIN2/CIN3/CIS

- with features suspicious for invasion (if invasion suspected)

• Squamous cell carcinoma

GLANDULAR CELL

• Atypical glandular cells of uncertain significance

• Atypical glandular cells- endocervical, endometrial NOS

• AGC, favor neoplastic

• Endocervical Adenocarcinoma in situ

• Adenocarcinoma

- endocervical

- endometrial

- extrauterine

- not otherwise specified (NOS)

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Statement of Specimen AdequacyStatement of Specimen Adequacy

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Adequate smearAdequate smearAn 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.

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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.

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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

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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.

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This specimen is unsatisfactory due to scant squamous cellularity seen at10X

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Cytomorphologic Criteria: Over 75% of cells are obscured by inflammation and blood.

.

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Defination and criteria for specimen Defination and criteria for specimen adequacyadequacy

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““Satisfacotry for evaluation”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.

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Satisfactory for evaluation but Satisfactory for evaluation but limited by….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.

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Unsatisfactory for evaluation…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.

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Any epithelial abnormality is of paramount importance and must be reported regardless of compromised specimen adequacy.

If abnormal cells are detected, the specimen is never categorised as “ UNSATISFACTORY”

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Infections Infections

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LACTOBACILLILACTOBACILLI

Lactobacilli are observed in about 50% of normal healthy adult female population.These bacilli release enzymes causing extensive cytolysis of glycogen containing cells.Mainly affect intermediate and superficial cells.Parabasal cells are generally spared. dr. monika nema

Bacterial vaginosisBacterial vaginosisBetween 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.

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Bacterial vaginosisBacterial vaginosis

Interpretation: NILM: Shift in Flora suggestive of bacterial vaginosis

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Herpes simplexHerpes simplex

Cytomorphologic Criteria: Nuclei showing "ground-glass" appearance. Multinucleation, nuclear molding, and dense eosinophilic intranuclear inclusions surrounded by a halo are also seen.

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ActinomycesActinomyces

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.

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CandidaCandida

Cytomorphologic Criteria: Pseudohyphae and reactive changes in the squamous epithelial cells.

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Trichomonas vaginalisTrichomonas vaginalis

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.

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Acute inflammationAcute inflammationDirty 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.

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INFLAMMATORY LESIONSINFLAMMATORY LESIONS ACUTE INFLAMMATIONACUTE INFLAMMATION

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ACUTE CERVICITISACUTE CERVICITIS

Polymorphs

nuclei with variable staining

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Lymphocytes

Plasma cells

CHRONIC CERVICITISCHRONIC CERVICITIS

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HyperkeratosisHyperkeratosis

Cytomorphologic Criteria: Anucleate mature polygonal squamous cells with ghost-like ?nuclear holes?

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Lymphocytic cervicitisLymphocytic cervicitis

Cytomorphologic Criteria: Polymorphous population of lymphoid cells and tingible body macrophages.

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REACTIVE CHANGES IN SQUAMOUS REACTIVE CHANGES IN SQUAMOUS CELLS IN INFLAMMATORY LESIONSCELLS IN INFLAMMATORY LESIONS

Nuclear pallor & enlargement

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INFLAMMATORY CHANGES IN INFLAMMATORY CHANGES IN ENDOCERVICAL CELLSENDOCERVICAL CELLSEnlargement,Presence of

prominent nucleoli

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INFLAMMATORY CHANGES IN INFLAMMATORY CHANGES IN ENDOCERVICAL CELLSENDOCERVICAL CELLSCytoplasmic

vacoulation of the endocervical cells

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IUD EFFECTIUD EFFECT

Calcified debris

Small cluster of glandular cells with cytoplasmic vacuoles displacing nuclei.creating a signet-ring appearance

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RADIATION EFFECTRADIATION EFFECT

Overall increase in cell sizeCytoplasmic vacuolation.Uneven staining of cytoplasm.Nuclear enlargement with vacuolation.Multinucleation.Fragmentation of nuclei.

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Endometrial cellsEndometrial cells

Squamous cells

Endometrial cells

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Epithelial cell abnormalityEpithelial cell abnormalityThe 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.

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ASC-USASC-USCells resemble superficial or

intermediate cells.Nuclear size is increased 2-3 times.Nuclear boundraies are regularor with

minimum irregularities.

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ASC-H (cannot exclude high grade ASC-H (cannot exclude high grade intraepithelial lesion)intraepithelial lesion)Cells resemble parabasal or basal in

configuration and size.Nuclei are hyperchromatic with uneven

chromatin pattern.Nuclear membrane is thick and uneven.

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ASCUS- MATUREASCUS- MATURE

Enlarged hyperchromatic nucleus

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ASC-H (cannot exclude high grade ASC-H (cannot exclude high grade intraepithelial lesion)intraepithelial lesion)

Loosely cohesive metaplastic cells with increased N:C ratio.

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ASC-HASC-Hmetaplastic cells and slightly enlarged nuclei with occasional nuclear contour irregularities.

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AGUSAGUS

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COMPARISON OF CLASSIFICATIONSCOMPARISON OF CLASSIFICATIONS

CIN CIN GRADEGRADE

WHOWHO BSCCBSCC BETHESDABETHESDA

borderlineborderline ASCUSASCUS

II Mild Mild dysplasiadysplasia

Mild Mild dyskaryosisdyskaryosis

Low grade Low grade SILSIL

IIII Moderate Moderate dysplasiadysplasia

Moderate Moderate dyskaryosisdyskaryosis

High grade High grade SILSIL

IIIIII Severe Severe dysplasiadysplasia

Severe Severe dyskaryosisdyskaryosis

High grade High grade SILSIL

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In CIN I and III , the characteristic nuclear changes are better observed in intermediate cells.

As the grades of CIN increase, parabasal and basal cells start showing nuclear abnormalities.

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Cervical intraepithelial neoplasia -1Cervical intraepithelial neoplasia -1

Slight nuclear enlargementHyperchromasia but finely granular chromatin.nuclear membrane is smooth and without indentation.

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Cervical intraepithelial Cervical intraepithelial neoplasia -IIneoplasia -II

Hyperchromasia is more marked.Chromatin can be seen in small clumps.Nuclear membrane is thickened slightly but indentation are ususlly absent.

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Cervical intraepithelial neoplasia Cervical intraepithelial neoplasia -III-III

Nuclear membrane is characteristically irregular and indented

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HPV infectionHPV infectionSeen in most of the cases of CIN.Lesions of CIN I and CIN II are usually

positive for HPV 6,11,31,42.Lesions of CIN III and invasive cancer are

usually positive for HPV 16,18,33.

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KOILOCYTE (HPV)KOILOCYTE (HPV)

Large squamous cell with enlarged hyperchromatic nucleus & large sharply demarcated perinuclear clear zone

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PSEUDOKOILOCYTESPSEUDOKOILOCYTES

Glycogen in squamous cells can give the appearance of "pseudokoilocytosis".

Nuclear abnormalities required for an interpretation of ASC-US/ LSIL are absent.

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Squamous cell carcinoma- non Squamous cell carcinoma- non keratinizingkeratinizing

Irregular chromatin, prominent nucleolus

Dysplastic squamous cells with anisocytosis and anisonucleosis including keratinization and tadpole cells are diagnostic of invasive squamous cell carcinoma.

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Squamous cell carcinom- Squamous cell carcinom- keratinizingkeratinizing

Tadpole cell

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Atypical endocervical cellsAtypical endocervical cells

enlarged round or oval nuclei with prominent nucleoli.

Mitotic figures

Sheet of cells with enlarged, variably-sized nuclei with some crowding and overlap of nuclei.

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Atypical columnar endocervical cells, with enlarged, elongated and hyperchromatic nuclei.

ENDOCERVICAL ADENOCARCINOMA ENDOCERVICAL ADENOCARCINOMA IN SITU (AIS)IN SITU (AIS)

Typical feathering and palisading.

Explanatory Notes: Pseudostratification, nuclear crowding and feathering are classic features of AIS dr. monika nema

ADENOCARCINOMAADENOCARCINOMA

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.

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Invasive adenocarcinoma Go back to the list

INVASIVE ADENOCARCINOMAINVASIVE ADENOCARCINOMA::

Cells are round,crowded,hyperchromatic

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INVASIVE ADENOCARCINOMAINVASIVE ADENOCARCINOMA

Crowding,large nucleoli

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ENDOMETRIAL ENDOMETRIAL ADENOCARCINOMAADENOCARCINOMA

Large hyperchromatic nucleus,Vacuolated cytoplasm

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Hormonal cytologyHormonal cytologyMaturation 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.

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Estrogens have proven action on 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.

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Cellular indices for hormonal Cellular indices for hormonal assessmentassessmentKaryopyknotic indexEosinophilic indexFolded cell indexCrowded cell indexMaturation index

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Karyopyknotic indexKaryopyknotic indexRatio 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.

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Eosinophilic indexEosinophilic indexRatio of mature squamous cells with

eosinophilic cytoplasm to all mature squamous cells irrespective of size of nucleus.

Peak value is 50-75 during ovulation.

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Folded cell indexFolded cell indexRatio of mature squamous cells with

folded margins to all mature squamous cells irrespective of staining chararcter.

Folding is usually observed in cells containing glycogen.

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Crowded cell indexCrowded cell indexRepresents the relationship of mature

squamous cells lying in clusters of four or more cells, to all mature squamous cells.

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Maturation indexMaturation indexIt is count of the parabasal cells,

intermediate and superficial cells .(P : I : S)In a normal menstruating woman during

ovulation the menstruation index will be 0/35/65.

In postmenopausal it will be 85/15/0.

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Maturation valueMaturation valueMeisels 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.

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Maturation value 100 – purely superficial cells. 0 – purely parabasal cells.50-95 – in normal menstruating woman.< 50 – atrophic squamous epithelium.

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DIFFRERENT PHASES OF MENSTRUAL CYCLEESTROGEN PHASE

eosinophilic superficial squamous cells, clean background.

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OVULATORY SMEAR

fern-like crystalline structures

eosinophilic superficial squamous cells

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. ( PROGESTERONE PHASE

basophilic squamous cells with folded cytoplasm (boat cells)

Glycogen deposits

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PROGESTERONE PHASE WITH ABUNDANT LACTOBACILLI AND CYTOLYSIS

navicular cells

Naked nuclei

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Menopause Menopause Maturation of cervical epithelium is

estrogen dependentDue to its insufficiency, maturation is

retarded.Number of mature squamous and

intermediate cells is reduced.In early menopause intermediate cells

increase in number.

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AtrophyAtrophy

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

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Amercian cancer society Amercian cancer society recommendationsrecommendations Screening should begin no 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, immunocompromised 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.

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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.

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THANK YOUTHANK YOU

SPEAKER : Dr. MONIKA NEMA

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