1

Squamous Lesions (SIL) Squamous Lesions (SIL)

including HPV and cancerincluding HPV and cancer

Dr. M. Desai Dr. M. Desai

Head of Cytopathology Service/Clinical Lead for Gynaecological CHead of Cytopathology Service/Clinical Lead for Gynaecological Cytology/Consultant Cytopathologist ytology/Consultant Cytopathologist

& Training School Director& Training School Director

Manchester Cytology Centre Manchester Cytology Centre

U.K.U.K.

TerminologyTerminology

�� BSCC (UK) current terminologyBSCC (UK) current terminology

�� BSCC (UK) proposed new terminologyBSCC (UK) proposed new terminology

�� Bethesda 2001 terminologyBethesda 2001 terminology

�� ECTP (European terminology)ECTP (European terminology)

1.1. Koilocytes (without Koilocytes (without

changes changes

suggestive of suggestive of

intraepithelial intraepithelial

neoplasia)neoplasia)

2.2. Squamous cell Squamous cell

changes (not changes (not

definitely definitely

neoplastic but neoplastic but

merit early merit early

repeat).repeat).

3.3. Atypical glandular Atypical glandular

cells (qualify).cells (qualify).

1.1. AtypicalAtypical

squamous cells ofsquamous cells of

(ASC(ASC--US) US)

undetermined undetermined

significance significance

2. ASC2. ASC--H (cannot H (cannot

exclude HSIL)exclude HSIL)

3. Atypical 3. Atypical

endocervical / endocervical /

endometrial / endometrial /

glandular cells: glandular cells:

NOS or favour NOS or favour

neoplastic. neoplastic.

1.1. Borderline change, Borderline change,

squamous, but not squamous, but not

otherwise specified. otherwise specified.

2.2. Borderline change, high Borderline change, high

grade dyskaryosis not grade dyskaryosis not

excluded.excluded.

3.3. Borderline change in Borderline change in

endocervical cells. endocervical cells.

Borderline nuclear change Borderline nuclear change

IncludeInclude

Unsatisfactory due to Unsatisfactory due to ……Unsatisfactory for Unsatisfactory for

evaluation evaluation Inadequate Inadequate Inadequate Inadequate

Within normal limits Within normal limits Negative for Negative for

intraepithelial lesion or intraepithelial lesion or

malignancy malignancy

Negative Negative Negative Negative

ECTP Terminology ECTP Terminology Bethesda system 2001 Bethesda system 2001 BSCC BSCC

Proposed new Proposed new

Terminology Terminology

BSCC 1986BSCC 1986

Comparison of BSCC (UK), Bethesda (USA) and ECTP (European) Terminology

From: Revised BSCC terminology : cytopathology Vol19 no.3 June 2008 P.137-157

ECTP Terminology ECTP Terminology Bethesda system 2001 Bethesda system 2001 BSCC BSCC

Proposed new Proposed new

Terminology Terminology

BSCC 1986BSCC 1986

Adenocarcinoma Adenocarcinoma

AISAIS

Endocervical Endocervical

Endometrial Endometrial

Extrauterine Extrauterine

NOS NOS

1.1. Endocervical Endocervical

carcinoma in situ carcinoma in situ

2.2. AdenocarcinomaAdenocarcinoma

EndocervicalEndocervical

EndometrialEndometrial

Extrauterine Extrauterine

NOSNOS

? Glandular neoplasia? Glandular neoplasia

Endocervical Endocervical

Non cervical Non cervical

? Glandular neoplasia ? Glandular neoplasia

1.1. Severe dysplasia Severe dysplasia

? Invasive.? Invasive.

2.2. Invasive Invasive

squamous cell squamous cell

carcinomacarcinoma

Squamous cell Squamous cell

carcinomacarcinomaSevere dyskaryosis ? Severe dyskaryosis ?

Invasive Invasive Severe dyskaryosis Severe dyskaryosis

? Invasive ? Invasive

1. Severe dysplasia 1. Severe dysplasia

(CIN3)(CIN3)

2. Carcinoma in situ 2. Carcinoma in situ

(CIN3)(CIN3)

HSILHSILSevere dyskaryosisSevere dyskaryosis

Moderate dysplasia Moderate dysplasia

(CIN2). (CIN2). HSILHSILHigh grade dyskaryosis High grade dyskaryosis Moderate dyskaryosis Moderate dyskaryosis

Mild dysplasiaMild dysplasia

(CIN1) (CIN1)

LSIL LSIL

Includes koilocytosisIncludes koilocytosis

Low grade dyskaryosis Low grade dyskaryosis

(includes all cases of (includes all cases of

koilocytosis provided that koilocytosis provided that

no high grade dyskaryosis no high grade dyskaryosis

is present)is present)

Mild dyskaryosis Mild dyskaryosis

Definition of Dyskaryosis Definition of Dyskaryosis

(SIL)(SIL)

�� Abnormal chromatin pattern is the essential Abnormal chromatin pattern is the essential requirement.requirement.

�� Finely speckled, punctate or coarsely clumped chromatinFinely speckled, punctate or coarsely clumped chromatin�� Abnormal size,spacing and distribution of chromatin Abnormal size,spacing and distribution of chromatin

granulesgranules�� Hyper or Hyper or HypochromasiaHypochromasia�� Irregular nuclear outline + or Irregular nuclear outline + or ––

�� Minor changes in nuclear outline e.g.single or multiple Minor changes in nuclear outline e.g.single or multiple folds,wrinkling or serration occur with inflammation and folds,wrinkling or serration occur with inflammation and degeneration.degeneration.

�� Large bulbous protrusions,scalloping or deep notches not Large bulbous protrusions,scalloping or deep notches not associated with linear folds occur in dyskaryosis.associated with linear folds occur in dyskaryosis.

�� Raised nuclear : cytoplasmic ratioRaised nuclear : cytoplasmic ratio

Normal Chromatin PatternNormal Chromatin Pattern

smooth with equal smooth with equal

distribution of nuclear chromatindistribution of nuclear chromatin

2

Abnormal Chromatin Pattern Abnormal Chromatin Pattern --

Finely SpeckledFinely Speckled

Abnormal Chromatin Pattern Abnormal Chromatin Pattern --

Finely Speckled and Punctate ChromatinFinely Speckled and Punctate Chromatin

Abnormal Chromatin Pattern Abnormal Chromatin Pattern -- Densely Densely

Hyperchromatic with Clumped ChromatinHyperchromatic with Clumped Chromatin

Definition of Dyskaryosis Definition of Dyskaryosis

(SIL)(SIL)�� Abnormal chromatin pattern is the essential Abnormal chromatin pattern is the essential requirement.requirement.

�� Finely speckled, punctate or coarsely clumped chromatinFinely speckled, punctate or coarsely clumped chromatin�� Abnormal size,spacing and distribution of chromatin Abnormal size,spacing and distribution of chromatin

granulesgranules�� Hyper or Hyper or HypochromasiaHypochromasia�� Irregular nuclear outline + or Irregular nuclear outline + or ––

�� Minor changes in nuclear outline e.g.single or multiple Minor changes in nuclear outline e.g.single or multiple folds,wrinkling or serration occur with inflammation and folds,wrinkling or serration occur with inflammation and degeneration.degeneration.

�� Large bulbous protrusions,scalloping or deep notches not Large bulbous protrusions,scalloping or deep notches not associated with linear folds occur in dyskaryosis.associated with linear folds occur in dyskaryosis.

�� raised nuclear : cytoplasmic ratioraised nuclear : cytoplasmic ratio

DYSKARYOTIC CELL (SIL)DYSKARYOTIC CELL (SIL)

Coarse chromatin with unequal Coarse chromatin with unequal

distributiondistribution

Abnormal Chromatin distributionAbnormal Chromatin distribution

3

Spot the DifferenceSpot the Difference

Fine,Speckled and coarse granularity but even distribution of

chromatin in NON-DYSKARYOTIC nuclei

Fine,speckled and coarse but irregularly distributed

chromatin of DYSKARYOTIC (SIL) nuclei

Definition of DyskaryosisDefinition of Dyskaryosis�� Abnormal chromatin pattern is the essential Abnormal chromatin pattern is the essential requirement.requirement.

�� Finely speckled, punctate or coarsely clumped chromatinFinely speckled, punctate or coarsely clumped chromatin�� Abnormal size,spacing and distribution of chromatin Abnormal size,spacing and distribution of chromatin

granulesgranules�� Hyper or Hyper or HypochromasiaHypochromasia

�� Irregular nuclear outline + or Irregular nuclear outline + or ––�� Minor changes in nuclear outline e.g.single or multiple Minor changes in nuclear outline e.g.single or multiple

folds,wrinkling or serration occur with inflammation and folds,wrinkling or serration occur with inflammation and degeneration.degeneration.

�� Large bulbous protrusions,scalloping or deep notches not Large bulbous protrusions,scalloping or deep notches not associated with linear folds occur in dyskaryosis. associated with linear folds occur in dyskaryosis.

�� Raised nuclear : cytoplasmic ratioRaised nuclear : cytoplasmic ratio

ChromasiaChromasia

��HyperchromasiaHyperchromasia

��HypochromasiaHypochromasia

Definition of Dyskaryosis (SIL)Definition of Dyskaryosis (SIL)

�� Abnormal chromatin pattern is the essential Abnormal chromatin pattern is the essential requirement.requirement.

�� Finely speckled, punctate or coarsely clumped chromatinFinely speckled, punctate or coarsely clumped chromatin�� Abnormal size,spacing and distribution of chromatin Abnormal size,spacing and distribution of chromatin

granulesgranules�� Hyper or Hyper or HypochromasiaHypochromasia

�� Irregular nuclear outline + or Irregular nuclear outline + or ––�� Minor changes in nuclear outline e.g.single or multiple Minor changes in nuclear outline e.g.single or multiple

folds,wrinkling or serration occur with inflammation and folds,wrinkling or serration occur with inflammation and degeneration.degeneration.

�� Large bulbous protrusions,scalloping or deep notches not Large bulbous protrusions,scalloping or deep notches not associated with linear folds occur in dyskaryosis. associated with linear folds occur in dyskaryosis.

�� Raised nuclear : cytoplasmic ratioRaised nuclear : cytoplasmic ratio

Nuclear folds in

normal cell

Minor changes in nuclear outline

in Inflammation

4

Abnormal Nuclear membrane Abnormal Nuclear membrane

In Dyskaryosis (SIL)In Dyskaryosis (SIL)

(Bulbous Protrusion)(Bulbous Protrusion)

Smooth nuclear membrane In

Dyskaryosis (SIL)

Spot the DifferenceSpot the Difference Spot the DifferenceSpot the Difference

Nuclear wrinkling and folding of

NON DYSKARYOTIC cell

Nuclear membrane protrusion

and deep notches in

DYSKARYOTIC (SIL) cell

Definition of Dyskaryosis (SIL)Definition of Dyskaryosis (SIL)

�� Abnormal chromatin pattern is the essential Abnormal chromatin pattern is the essential requirement.requirement.

�� Finely speckled, punctate or coarsely clumped chromatinFinely speckled, punctate or coarsely clumped chromatin�� Abnormal size,spacing and distribution of chromatin Abnormal size,spacing and distribution of chromatin

granulesgranules�� Pale vs. HyperchromaticPale vs. Hyperchromatic

�� Irregular nuclear outline + or Irregular nuclear outline + or ––�� Minor changes in nuclear outline e.g.single or multiple Minor changes in nuclear outline e.g.single or multiple

folds,wrinkling or serration occur with inflammation and folds,wrinkling or serration occur with inflammation and degeneration.degeneration.

�� Large bulbous protrusions,scalloping or deep notches not Large bulbous protrusions,scalloping or deep notches not associated with linear folds occur in dyskaryosis. associated with linear folds occur in dyskaryosis.

�� Raised nuclear : cytoplasmic ratioRaised nuclear : cytoplasmic ratio

Raised Nuclear:Cytoplasmic RatioRaised Nuclear:Cytoplasmic Ratio

�� Used for grading of dyskaryosis (SIL)Used for grading of dyskaryosis (SIL)

�� Nucleus occupies more than 50% of area of Nucleus occupies more than 50% of area of

cytoplasm in High Grade Dyskaryosis (HSIL)cytoplasm in High Grade Dyskaryosis (HSIL)

�� High Grade dyskaryosis (HSIL) is found in less High Grade dyskaryosis (HSIL) is found in less

mature cellsmature cells

�� Moderate dyskaryosis is seen in larger and Moderate dyskaryosis is seen in larger and

intermediate size parabasal squamous cellsintermediate size parabasal squamous cells

�� Severe dyskaryosis is seen in small parabasal Severe dyskaryosis is seen in small parabasal

and basal squamous cellsand basal squamous cells

5

Moderate Dyskaryosis Severe Dyskaryosis

HSIL HSIL

Pitfalls (false positive) Pitfalls (false positive) -- high high

nuclear:cytoplasmicnuclear:cytoplasmic ratio with:ratio with:

��Endocervical cellsEndocervical cells

��Immature squamous metaplastic Immature squamous metaplastic

cellscells

��Endometrial cellsEndometrial cells

��LymphocytesLymphocytes

Endometrials

Follicular cervicitis Endocervicals

Immature metaplastics Mild Dyskaryosis (LSIL)Mild Dyskaryosis (LSIL)Back to BasicsBack to Basics

�� Cells occur singly and in sheetsCells occur singly and in sheets

�� Nuclear changes in mature superficial or Nuclear changes in mature superficial or intermediate cellsintermediate cells

�� Nuclear enlargementNuclear enlargement

�� mild dyskaryotic (LSIL) nuclei at least x3 size mild dyskaryotic (LSIL) nuclei at least x3 size of intermediate nucleusof intermediate nucleus

�� Chromatin irregularityChromatin irregularity

�� Irregularity of nuclear membrane + or Irregularity of nuclear membrane + or --

–– NotchesNotches

–– ProtrusionsProtrusions

–– SpursSpurs–– Not just folds or wavy outlineNot just folds or wavy outline

Mild Dyskaryosis (LSIL)Mild Dyskaryosis (LSIL)Back To BasicsBack To Basics

�� Dyskaryotic nucleusDyskaryotic nucleus

�� Enlarged nucleus <50% of cell areaEnlarged nucleus <50% of cell area

�� Bi and multinucleation often seenBi and multinucleation often seen

�� Polygonal cytoplasm of mature Polygonal cytoplasm of mature

squamous cells, individual cell squamous cells, individual cell

borders borders

�� Strong association withStrong association with

koilocytosis koilocytosis

ASCUS or LSIL? LSIL or HSIL?

Spot the difference

6

Borderline(ASCUS)

TO

Mild Dyskaryosis (LSIL)

Mild to Moderate Dyskaryosis

(LSIL TO HSIL)

Subjectivity Still Persist : Not a full proof Science

Dyskaryosis (SIL)

? Grade

Spot The Spot The

DifferenceDifferenceSpot The Spot The

DifferenceDifference

Mild Dyskaryosis (LSIL)

Borderline changes (ASCUS)

Features of highFeatures of high--grade dyskaryosisgrade dyskaryosis

(HSIL)(HSIL)Back To BasicsBack To Basics

�� Increased depth of focus Increased depth of focus

�� Irregularity of nuclear membrane (walnut) Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small abnormal Increase in single, isolated and small abnormal cellscells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty spaces Isolated abnormal cells present in empty spaces between cell clustersbetween cell clusters

�� Rounding up of cells resulting in smaller size and Rounding up of cells resulting in smaller size and higher N:C ratiohigher N:C ratio

�� Cleaner backgroundCleaner background

7

Features of highFeatures of high--grade dyskaryosisgrade dyskaryosis

(HSIL)(HSIL)Back To BasicsBack To Basics

�� Increased depth of focus Increased depth of focus

�� Irregularity of nuclear membrane (walnut)Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small abnormal Increase in single, isolated and small abnormal cellscells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty spaces Isolated abnormal cells present in empty spaces between cell clustersbetween cell clusters

�� Rounding up of cells resulting in smaller size and Rounding up of cells resulting in smaller size and higher N:C ratiohigher N:C ratio

�� Cleaner backgroundCleaner background

Features of highFeatures of high--grade dyskaryosis grade dyskaryosis

(HSIL)(HSIL)

�� Increased depth of focus Increased depth of focus

�� Irregularity of nuclear membrane (walnut)Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small Increase in single, isolated and small abnormal cellsabnormal cells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty spaces Isolated abnormal cells present in empty spaces between cell clustersbetween cell clusters

�� Rounding up of cells resulting in smaller size and Rounding up of cells resulting in smaller size and higher N:C ratiohigher N:C ratio

�� Cleaner backgroundCleaner background

8

Features of highFeatures of high--grade dyskaryosis grade dyskaryosis

(HSIL)(HSIL)

�� Increased depth of focus Increased depth of focus

�� Irregularity of nuclear membrane (walnut) Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small abnormal Increase in single, isolated and small abnormal cellscells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty spaces Isolated abnormal cells present in empty spaces between cell clustersbetween cell clusters

�� Rounding up of cells resulting in smaller size and Rounding up of cells resulting in smaller size and higher N:C ratiohigher N:C ratio

�� Cleaner backgroundCleaner background

Features of highFeatures of high--grade dyskaryosis grade dyskaryosis

(HSIL)(HSIL)

�� Increased depth of focus Increased depth of focus

�� Irregularity of nuclear membrane (walnut) Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small abnormal Increase in single, isolated and small abnormal cellscells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty Isolated abnormal cells present in empty spaces between cell clusters in LBC and in spaces between cell clusters in LBC and in streaks in conventional smearsstreaks in conventional smears

�� Rounding up of cells resulting in smaller size and Rounding up of cells resulting in smaller size and higher N:C ratiohigher N:C ratio

�� Cleaner backgroundCleaner background

Features of highFeatures of high--grade dyskaryosis grade dyskaryosis

(HSIL)(HSIL)

�� Increased depth of focus Increased depth of focus

�� Irregularity of nuclear membrane (walnut) Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small abnormal Increase in single, isolated and small abnormal cellscells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty spaces Isolated abnormal cells present in empty spaces between cell clustersbetween cell clusters

�� Rounding up of cells resulting in smaller size Rounding up of cells resulting in smaller size and higher N:C ratioand higher N:C ratio

�� Cleaner backgroundCleaner background

9

Features of highFeatures of high--grade dyskaryosis grade dyskaryosis

(HSIL)(HSIL)

�� Increased depth of focusIncreased depth of focus

�� Irregularity of nuclear membrane (walnut) Irregularity of nuclear membrane (walnut)

�� Increase in single, isolated and small abnormal Increase in single, isolated and small abnormal cellscells

�� Few abnormal cellsFew abnormal cells

�� Isolated abnormal cells present in empty spaces Isolated abnormal cells present in empty spaces between cell clustersbetween cell clusters

�� Rounding up of cells resulting in smaller size and Rounding up of cells resulting in smaller size and higher N:C ratiohigher N:C ratio

�� Cleaner background in LBCCleaner background in LBC

Grading of dyskaryosis (SIL)Grading of dyskaryosis (SIL)Useful TipsUseful Tips

�� Higher N:C ratios / areas / diameter with Higher N:C ratios / areas / diameter with increasing grade of dyskaryosis (SIL).increasing grade of dyskaryosis (SIL).

�� HSIL (Moderate between HSIL (Moderate between ½½ to 2/3 to 2/3

Severe > 2/3)Severe > 2/3)

�� Mild (LSIL) in superficial and Intermediate cells Mild (LSIL) in superficial and Intermediate cells

�� HSIL( Moderate in Large and intermediate size HSIL( Moderate in Large and intermediate size parabasal and Severe in Small parabasal and parabasal and Severe in Small parabasal and basal cells.)basal cells.)

�� Dyskaryosis (SIL) in immature squamous cells Dyskaryosis (SIL) in immature squamous cells most likely represents highmost likely represents high--grade dyskaryosis grade dyskaryosis (HSIL)(HSIL)

Grade the Dyskaryosis (SIL) ?

10

Moderate and Severe

Dyskaryosis

(HSIL)

Mild and Moderate

Dyskaryosis

(LSIL AND HSIL)

Grade the Dyskaryosis (SIL) ?Grade the Dyskaryosis (SIL)

Grade the Dyskaryosis (SIL)

Mild Dyskaryosis/LSIL

Grade the Dyskaryosis (SIL)

Grade the Dyskaryosis (SIL)

Moderate Dyskaryosis/HSIL

Grade the Dyskaryosis (SIL)

11

Grade the Dyskaryosis (SIL)

Severe Dyskaryosis/HSIL

Grade the Dyskaryosis (SIL)

Grade the Dyskaryosis (SIL)

Low grade or High Grade?

Subjectivity persist

Grade the Dyskaryosis (SIL)

Grade the Dyskaryosis (SIL)

Low Grade or High Grade?

Subjectivity persist

Patterns of Dyskaryosis (SIL)Patterns of Dyskaryosis (SIL)

Pattern recognition and experiencePattern recognition and experience

12

Patterns of dyskaryosis (SIL)Patterns of dyskaryosis (SIL)

�� Ungraded Dyskaryosis (SIL)Ungraded Dyskaryosis (SIL)

�� Large cell nonLarge cell non--keratinising (keratinising (SyncitialSyncitial))

�� KeratinisingKeratinising

�� MixedMixed

�� Scanty dyskaryosis (SIL)Scanty dyskaryosis (SIL)

�� Small cell dyskaryosis (SIL)Small cell dyskaryosis (SIL)

�� CIN microbiopsies (CIN microbiopsies (HCGsHCGs))

�� Flag cells Flag cells

�� Pale cell dyskaryosis (SIL)Pale cell dyskaryosis (SIL)

�� Bland cell dyskaryosis (SIL) In Bland cell dyskaryosis (SIL) In ThinprepThinprep

Ungraded Dyskaryosis Ungraded Dyskaryosis

(SIL)(SIL)

�� Dyskaryotic bare nucleiDyskaryotic bare nuclei

�� Loss of cytoplasm due to cytolysis or atrophyLoss of cytoplasm due to cytolysis or atrophy

�� Loss of cell boundariesLoss of cell boundaries

�� Grade as High grade dyskaryosis (HSIL)Grade as High grade dyskaryosis (HSIL)

High Grade Dyskaryosis with

bare nulcei

Ungraded Dyskaryosis

Large cell nonLarge cell non--keratinising (keratinising (syncitialsyncitial))

High High gradeDyskaryosisgradeDyskaryosis (HSIL)(HSIL)

�� Loose syncytial groupingsLoose syncytial groupings

�� Nuclei four to six times diameter of Nuclei four to six times diameter of polymorphspolymorphs

�� Nuclei rounded and pale stainingNuclei rounded and pale staining

�� Finely speckled chromatinFinely speckled chromatin

�� Cytoplasm relatively abundant, delicate and Cytoplasm relatively abundant, delicate and indistinctindistinct

�� N/C ratio difficult to assess, usually less than N/C ratio difficult to assess, usually less than half, misinterpreted as mild half, misinterpreted as mild diskaryosisdiskaryosis (LSIL)(LSIL)

�� Misinterpreted as CGIN when in continuity Misinterpreted as CGIN when in continuity with endocervical cellswith endocervical cells

Large cell non-keratinising severe dyskaryosis

Syncytial variety

Keratinising High grade Keratinising High grade

Dyskaryosis (HSIL)Dyskaryosis (HSIL)

�� Cytologically indistinguishable from Cytologically indistinguishable from

keratinising squamous cell carcinomakeratinising squamous cell carcinoma

13

Small Cell Dyskaryosis Small Cell Dyskaryosis

(HSIL)(HSIL)

�� Monotonous cell populationMonotonous cell population

�� Abnormal chromatin patternAbnormal chromatin pattern

�� Nuclear diameter less than twice of Nuclear diameter less than twice of polymorphs or lymphocytepolymorphs or lymphocyte

�� High High nuclear:cytoplasmicnuclear:cytoplasmic ratioratio

�� Can have regular nuclear membraneCan have regular nuclear membrane

�� D/D immature metaplastic D/D immature metaplastic cell,EM,Histiocyte,Follicularcell,EM,Histiocyte,Follicularcervicitis,atrophiccervicitis,atrophic EC and atrophic parabasal EC and atrophic parabasal squamous cellssquamous cells

Small cell dyskaryosisSmall cell dyskaryosisScreening and Interpretation ErrorScreening and Interpretation Error

Small cell dyskaryosisSmall cell dyskaryosis

Misinterpreted asMisinterpreted as

�� Normal endometrial cellNormal endometrial cell

�� Immature metaplastic cellsImmature metaplastic cells

�� LymphocyteLymphocyte

�� Follicular cervicitisFollicular cervicitis

�� Normal HistiocyteNormal Histiocyte

�� Atrophic endocervical cellsAtrophic endocervical cells

�� Normal parabasal squamous cellNormal parabasal squamous cell

HISTIOCYTES,Small cell severe dyskaryosis

LYMPHOCYTES

FOLLICULAR CERVICITIS

ENDOMETRIALS

Sparse (Scanty) dyskaryosis (SIL)Sparse (Scanty) dyskaryosis (SIL)

�� Litigation cellLitigation cell

�� Easily overlooked in routine screeningEasily overlooked in routine screening

�� More difficult to recognise if pale,small and More difficult to recognise if pale,small and

singlesingle

14

Scanty Dyskaryosis (SIL): overlapping of the

fields is essential in screening

Scanty dyskaryosis

Litigation cell

Scanty Dyskaryosis (SIL) Flag CellFlag Cell

�� Small keratinised cell with pyknotic, Small keratinised cell with pyknotic,

India ink nucleusIndia ink nucleus

�� On its own should not be reported as On its own should not be reported as

High grade dyskaryosisHigh grade dyskaryosis

�� Search for High grade dyskaryosis in Search for High grade dyskaryosis in

other areasother areas

Flag cells : Search for High grade dyskaryosis

(HSIL) or invasive features

Flag cell Associated High grade dyskaryosis

15

Pale cell Dyskaryosis (SIL)Pale cell Dyskaryosis (SIL)

�� Hypo chromatic nucleiHypo chromatic nuclei

�� May be a staining artefactMay be a staining artefact

�� Identified by abnormal chromatin Identified by abnormal chromatin

patternpattern

�� Can be seen in all grades of Can be seen in all grades of

dyskaryosis (SIL) and types of cellsdyskaryosis (SIL) and types of cells

�� Often present mixed with some Often present mixed with some

““normalnormal”” staining cells staining cells

�� Nuclear membrane irregularity + or Nuclear membrane irregularity + or --

Pale cell Dyskaryosis (SIL)Pale cell Dyskaryosis (SIL)

Pale dyskaryosis /SILPale dyskaryosis /SIL Pale dyskaryosis/SILPale dyskaryosis/SIL

Pale dyskaryosis/SILPale dyskaryosis/SIL

Bland Dyskaryosis/SIL: Bland Dyskaryosis/SIL:

A New Pitfall in Liquid Based A New Pitfall in Liquid Based

Cytology (ThinPrep)Cytology (ThinPrep)

Dr. Dr. D.N.Rana,Dr.K.DentonD.N.Rana,Dr.K.Denton,,

Dr. M. Desai & Dr. M. LynchDr. M. Desai & Dr. M. LynchCytopathology MayCytopathology May--June 2008,June 2008,

19(3) P16219(3) P162--166166

16

Bland Dyskaryosis (SIL)Bland Dyskaryosis (SIL)

�� Dyskaryotic/SIL cells appear deceptively bland Dyskaryotic/SIL cells appear deceptively bland

on low on low -- power examinationpower examination

�� Misinterpreted as squamous metaplastic cells Misinterpreted as squamous metaplastic cells

or endocervical cellsor endocervical cells

�� Seen in groupsSeen in groups

�� Disorganised architecture within the groupDisorganised architecture within the group

BLAND CELL DYSKARYOSIS (SIL)

•Chaotic group

• Mimicking endocervicals

Bland Dyskaryosis (SIL)Bland Dyskaryosis (SIL)�� Variation in cell sizeVariation in cell size

�� Raised nuclear:cytoplasmic ratioRaised nuclear:cytoplasmic ratio

�� Speckled / punctate chromatin patternSpeckled / punctate chromatin pattern

�� Chromatin may be evenly distributedChromatin may be evenly distributed

�� Smooth nuclear outlinesSmooth nuclear outlines

Bland cell dyskaryosis (SIL)Overcrowding

• Speckled chromatin pattern

• Smooth nuclear membranes

CIN Microbiopsies (CIN Microbiopsies (HCGsHCGs))�� 3D groups 3D groups

�� Nuclear crowding Nuclear crowding

�� Disorderly chaotic architectureDisorderly chaotic architecture

�� Steep relief at edge of groupSteep relief at edge of group

�� Evaluate the cells at edgeEvaluate the cells at edge

�� Abnormal nuclear chromatinAbnormal nuclear chromatin

�� Hyperchromatic nucleiHyperchromatic nuclei

�� Nuclear membrane irregularities less pronounced Nuclear membrane irregularities less pronounced

�� Increased mitotic activity Increased mitotic activity

�� Nucleoli more visibleNucleoli more visible

17

ALLALL of the following cells are of the following cells are

normal !normal !

ALL ALL of the following cells are of the following cells are

abnormal !abnormal !

18

HighHigh--grade dyskaryosis (HSIL)grade dyskaryosis (HSIL)

PitfallsPitfalls

�� Metaplastic cellsMetaplastic cells

�� Endometrial cellsEndometrial cells

�� HistiocytesHistiocytes

�� Atrophy Atrophy

�� Blue blobsBlue blobs

�� IUCD cellsIUCD cells

�� Reactive inflammatory sheetsReactive inflammatory sheets

�� Glandular neoplasia (cervical type)Glandular neoplasia (cervical type)

Invasive Squamous Invasive Squamous

CarcinomaCarcinoma

Different types of Invasive Different types of Invasive

Squamous Cell CarcinomaSquamous Cell Carcinoma

�� Micro invasiveMicro invasive

�� Keratinising Keratinising

�� Non keratinising large cellNon keratinising large cell

�� Non keratinising small cellNon keratinising small cell

�� VerrucousVerrucous carcinomacarcinoma

MicroinvasiveMicroinvasive

* * Can not be diagnosed reliablyCan not be diagnosed reliably

* Indistinguishable from severe * Indistinguishable from severe

dyskaryosisdyskaryosis

* Macronucleoli in * Macronucleoli in syncitialsyncitial

aggregates of severe dyskaryosisaggregates of severe dyskaryosis

* Irregular chromatin distribution* Irregular chromatin distribution

19

Invasive Squamous cellInvasive Squamous cell

Carcinoma: Carcinoma:

Architectural featuresArchitectural features

* Tumour diathesis* Tumour diathesis

* Large numbers of dyskaryotic cells* Large numbers of dyskaryotic cells

(severe dyskaryosis)(severe dyskaryosis)

* Single cells, loose aggregate* Single cells, loose aggregate

* Sheets of undifferentiated cells * Sheets of undifferentiated cells

HCCGHCCG

Invasive squamous cellInvasive squamous cell

carcinoma :carcinoma :

Cellular featuresCellular features

* Very bizarre cell shapes* Very bizarre cell shapes

* Fibre and tadpole cells* Fibre and tadpole cells

* Cell embracement ( cell into cell * Cell embracement ( cell into cell

pattern)pattern)

Invasive Squamous cellInvasive Squamous cell

Carcinoma:Carcinoma:

Nuclear featuresNuclear features

•Marked pleomorphism

( Different size & shape)

• Irregular Nuclear Membrane

• Coarse, granular chromatin patternCoarse, granular chromatin pattern

• Hyperchromasia with windowing

(parachromatin clearing)

•• Angulated chromatinAngulated chromatin

•• Mitotic figureMitotic figure

•• irregular macronucleoliirregular macronucleoli

Tumour DiathesisTumour Diathesis

**Grey/blue granular material Grey/blue granular material

clinging to malignant cellsclinging to malignant cells

* Blood fresh and old (* Blood fresh and old (haemosiderinehaemosiderine))

* Fibrin and protein* Fibrin and protein

* * Cell necrosis and debrisCell necrosis and debris

* Nuclear and cytoplasmic fragment* Nuclear and cytoplasmic fragment

* * polyspolys ,inflammatory dirty background,inflammatory dirty backgroundDiathesis is rare, reflects ulceration of surface mucosa and Diathesis is rare, reflects ulceration of surface mucosa and

associated with late stage disease associated with late stage disease

Absent in Absent in microinvasivemicroinvasive and and verrucousverrucous carcinomacarcinoma

Benign DiathesisBenign Diathesis

*Inflammatory,dirty background*Inflammatory,dirty background

*Absence of Malignant cells*Absence of Malignant cells

*Severe infection e.g.Trichomonas*Severe infection e.g.Trichomonas

*Severe atrophy*Severe atrophy

*Abscess*Abscess

*Necrotic,ulcerated polyp*Necrotic,ulcerated polyp

Tumour DiathesisTumour Diathesis

Benign DiathesisBenign Diathesis

20

Abnormal nucleoliAbnormal nucleoli

�� These can be single or multipleThese can be single or multiple

�� If single, they are usually large and If single, they are usually large and

abnormal with windowingabnormal with windowing

�� If multiple the nucleoli vary in size and If multiple the nucleoli vary in size and

shapeshape

�� They may be red stainingThey may be red staining

NucleoliNucleoli

NucleoliNucleoli NucleoliNucleoli

Malignant Malignant ReactiveReactive

Small Cell NonSmall Cell Non--keratinizingkeratinizing

Squamous CarcinomaSquamous Carcinoma

* Tumour cells occur singly or in small

discohesive syncitial like groups.

* Cells small, cuboidal or round

* Scanty cytoplasm

* High N/C ratio

* No keratinization

* Nuclei large, round to oval

* Hyperchromatic, coarsley granulated chromatin

* Nucleoli small, may be obscured

Differential Diagnosis of Small Cell Differential Diagnosis of Small Cell

Squamous cell carcinomaSquamous cell carcinoma

* Normal and abnormal endometrial cells

* Lymphoid lesions

* Small cell severe dyskaryosis

* Reserve cell hyperplasia

21

Large CellLarge Cell

NonNon--KeratinizingKeratinizing

Squamous cell carcinomaSquamous cell carcinoma

* Numerous single cells and discohesive

syncitial groups of cells

* Cellular and nuclear pleomorphism is

less than keratinizing SCC

* High N/C ratio

* Nuclear overlapping

Differential Diagnosis of Large cell, NonDifferential Diagnosis of Large cell, Non--

keratinizing Squamous cell carcinomakeratinizing Squamous cell carcinoma

* * Repair cellsRepair cells

* * Endocervical AdenocarcinomaEndocervical Adenocarcinoma

Repair CellsRepair Cells

* Cohesive, flat, monolayered sheets of polygonal

squamous cells

* Distinct cytoplasmic boundaries

* Frequent cytoplasmic projectious (pseudopodia)

* Tumour diathesis ABSENT

cont……

Repair CellsRepair Cells

* Inflammatory benign diathesis

* Cytoplasm delicate, cyanophilic, no keratinization

* Cytoplasmic vacuoles containing neutrophils and nuclear

debris

* Nuclei enlarged

* N/C ratio is only slightly or moderately Increased

* Nuclear chromatin finely granular and evenly distributed

* One or several prominent eosinophlilic nucleoli

* Mitotic figures may be present

Differential Diagnosis of

Repair Cells vs Non-keratinizing SCC

Features Favour Repair

* Flat sheet arrangement of cells rather than

syncitia or three dimensional clusters

* Lack of nuclear overlapping

* Lack or rarity of single cells

* Lack of tumour diathesis

* Fine Chromatin

22

Quiz TimeQuiz Time

Atypical repairAtypical repair

Invasive CarcinomaInvasive Carcinoma

Keratinizing Keratinizing

Squamous Cell CarcinomaSquamous Cell Carcinoma

* Highly discohesive cells

* Spindle keratinized cells* Keratinised cells with bizarre cytoplasmic

forms

* Tadpole and caudate Cells* Nuclei hyperchromatic with coarsely clumped,

irregularly distributed chromatin

* Indistinct nucleoli

* Tumour diathesis maybe absent

Differential Diagnosis Differential Diagnosis

of Keratinizing SCCof Keratinizing SCC

Atypical parakeratosis/ dyskeratosis

- individual cell may look malignant but are

too small

- can occur in Sq.cell ca, inflammation,

condyloma (wart) and keratinizing CIN

Keratinizing SCCKeratinizing SCC

False negative resultFalse negative result

*Infected,ulcerated tumour*Infected,ulcerated tumour

*Scanty malignant cells*Scanty malignant cells

*Verrucous carcinoma*Verrucous carcinoma

VerrucousVerrucous

CarcinomaCarcinoma

*Rare tumour*Rare tumour

*Slow growing, *Slow growing, exophyticexophytic tumour,tumour,

*rarely metastasize*rarely metastasize

*Cytology misleading*Cytology misleading

*Hyperkeratosis and *Hyperkeratosis and parakeratosisparakeratosis

*Scanty *Scanty dyskaryoticdyskaryotic cellscells

*Absence of Malignant nuclei*Absence of Malignant nuclei

23

AcknowledgementAcknowledgement

My special thanks to My special thanks to

Dr. Dr. Rana,DrRana,Dr. Denton & Dr. . Denton & Dr. BijalBijal Shah Shah

From UKFrom UK

&&

Mr.Andrew Mr.Andrew EveredEvered

From Wales Training SchoolFrom Wales Training School

ForFor

Producing images and drawingsProducing images and drawings

andand

Writing some of the textsWriting some of the texts

I have also used I have also used HologicHologic CytycCytyc ThinprepThinprep Images, BD Images, BD SurepathSurepath Images Images from NHSCSP atlas and images Images Images from NHSCSP atlas and images and the tables from arts and Science of cytology book from and the tables from arts and Science of cytology book from Prof. Prof. DeMayDeMay for this presentationfor this presentation

Cytology of HPVCytology of HPV

Two Two PathognomicPathognomic cell patterncell pattern

�� KoilocyteKoilocyte

�� DyskeratocyteDyskeratocyte

Other NonOther Non-- specific cell patternsspecific cell patterns

�� Cells Cells –– MacrocytesMacrocytes , Kites, balloons, Kites, balloons

�� Cytoplasm Cytoplasm –– Polka dots, cracksPolka dots, cracks

�� Nuclei Nuclei –– Bi and Bi and multinucleationmultinucleation, spindling, , spindling, smudgingsmudging

KoilocytesKoilocytes

�� KoiloKoilo = Hollow or cavity= Hollow or cavity�� CytopathicCytopathic effect of HPV infectioneffect of HPV infection�� Intermediate or superficial Intermediate or superficial squamoussquamous cells cells containing large containing large perinuclearperinuclear cavity with cavity with sharply cut borderssharply cut borders

�� Dense Dense eosinophiliceosinophilic or or cyanophiliccyanophilic cytoplasm cytoplasm peripheral to the cavityperipheral to the cavity

�� Enlarged abnormal nucleus Enlarged abnormal nucleus �� Chromatin varies from degenerate (smudged Chromatin varies from degenerate (smudged pyknoticpyknotic, fragmented) to normal or slight , fragmented) to normal or slight coarsening coarsening

�� Nuclear membrane NOT apparentNuclear membrane NOT apparent�� Inconspicuous nucleoliInconspicuous nucleoli

�� No inclusion bodiesNo inclusion bodies�� Bi and Bi and MultinucleationMultinucleation is common is common

KoilocytoticKoilocytotic AtypiaAtypiaBack To BasicsBack To Basics

�� Clear well defined area around Clear well defined area around

nucleusnucleus

�� Thickened edge, sharply Thickened edge, sharply

demarcated borderdemarcated border

�� Some nuclear Some nuclear atypiaatypia

Group of Koilocytes

24

Diagnostic DilemmasDiagnostic Dilemmas

Koilocyte Simulation (Koilocyte Simulation (PseudokoilocytePseudokoilocyte))

�� Non HPV changes Non HPV changes

�� Rolling edge of cytoplasm (common with LBC)Rolling edge of cytoplasm (common with LBC)

�� Intracytoplasmic glycogen lacking yellowIntracytoplasmic glycogen lacking yellow--brown brown stainingstaining

�� Non HPV inflammationNon HPV inflammation

TerminologyTerminology

�� USAUSA--Bethesda Bethesda –– LSILLSIL

�� UK UK –– BL vs. Mild BL vs. Mild dyskdysk

�� ECTPECTP--EuropeanEuropean--Separate categorySeparate category

Figure 162 Figure 163 Figure 164

Figure 165 Figure 166

Koilocyte Simulation

Diagnostic DilemmaDiagnostic Dilemma

TerminologyTerminology

Theories of pathogenesisTheories of pathogenesis

�� KoilocyteKoilocyte is cellular changes of HPV infection is cellular changes of HPV infection that mimics dysplasia but is not genuine that mimics dysplasia but is not genuine dysplasiadysplasia

�� KoilocyteKoilocyte is precursor to dysplasiais precursor to dysplasia

�� KoilocyteKoilocyte is dysplasiais dysplasia

Current TerminologiesCurrent Terminologies

�� USAUSA-- BethesdaBethesda--KoilocyteKoilocyte is dysplasiais dysplasia = = LSILLSIL

�� UKUK-- KoilocyteKoilocyte is precursor to dysplasiais precursor to dysplasia ==

BNC or mild BNC or mild dyskaryosisdyskaryosis dependent dependent on nuclear abnormalityon nuclear abnormality

�� ECTP ECTP –– koilocytekoilocyte is cellular changes of is cellular changes of infectioninfection= separate entity= separate entity

KoilocyteKoilocyte TerminologyTerminology

U TurnU Turn

Bethesda Explanatory NotesBethesda Explanatory Notes

�� KoilocyticKoilocytic nuclear changes that fall short of nuclear changes that fall short of a definitive SIL interpretation may be a definitive SIL interpretation may be categorised as ASCcategorised as ASC--USUS

Revised BSCC (UK) TerminologyRevised BSCC (UK) Terminology

�� In In koilocytosiskoilocytosis, it is difficult to achieve , it is difficult to achieve consistency in the differentiation of nuclear consistency in the differentiation of nuclear changes between BNC and Mild changes between BNC and Mild DyskaryosisDyskaryosis, , therefore combine them as Lowtherefore combine them as Low--grade grade squamoussquamous dyskaryosisdyskaryosis

BNC vs. Mild dysk ASCUS vs. LSIL

T

E

R

M

I

N

O

L

O

G

Y

Spot The Spot The

DifferenceDifference

25

HPV CytologyHPV Cytology

DyskeratocytesDyskeratocytes

�� Abnormal Abnormal KeratinizationKeratinization

�� Keratinized squamous cells staining Keratinized squamous cells staining brilliant orange with OGbrilliant orange with OG--6 of pap stain6 of pap stain

( Pink with ( Pink with SurepathSurepath LBC )LBC )

�� Single or in thick, 3D clusters Single or in thick, 3D clusters

�� Clusters without discernible polarityClusters without discernible polarity

�� Nuclear enlargement with condensed or Nuclear enlargement with condensed or granular chromatin detailsgranular chromatin details

DyskeratocytesDyskeratocytes

In The Absence Of HPV In The Absence Of HPV

InfectionInfection�� Small keratinized pearls, rafts or spikesSmall keratinized pearls, rafts or spikes

�� Cells arranged in orderly pattern Cells arranged in orderly pattern

�� Small,uniform,widely spaced nuclei that Small,uniform,widely spaced nuclei that

flattens towards the edge to lie along the flattens towards the edge to lie along the

long axis of the grouplong axis of the group

�� No nuclear abnormalityNo nuclear abnormality

Dyskeratotic cells Dyskeratotic cells

Without HPV With HPV

Other NonOther Non--specific cell specific cell

patterspatters

Macrocyte Kite Cells

Balloon Cells Polka Dot

Incidence of HPV InfectionIncidence of HPV Infection

�� Incidence 4 % by cytology diagnosisIncidence 4 % by cytology diagnosis

and 20and 20--30% by molecular diagnosis 30% by molecular diagnosis

�� KoilocytesKoilocytes are covered by are covered by dyskeratoticdyskeratotic cells cells

and not shedding in many cases of HPV and not shedding in many cases of HPV

infectioninfection

Cytology severely underestimates the true Cytology severely underestimates the true

incidence of HPV infectionincidence of HPV infection

Cytology of HPV infectionCytology of HPV infection

After HPV TestAfter HPV Test

�� Cytology Cytology Negative Negative / HPV positive/ HPV positive

�� Cytology BNC with Cytology BNC with KoilocyteKoilocyte (LSIL)(LSIL) / /

HPV positiveHPV positive

�� Cytology BNC without Cytology BNC without koilocytekoilocyte

(ASCUS)(ASCUS) / HPV positive/ HPV positive

�� Cytology Cytology DyskaryosisDyskaryosis (LSIL OR HSIL)/(LSIL OR HSIL)/

HPV positiveHPV positive

26

Cytology reporting Cytology reporting

after HPV Testafter HPV Test

Should we be reporting Should we be reporting KoilocyteKoilocyte

in cytology?in cytology?

�� KoilocyteKoilocyte demonstrates complete demonstrates complete

infective viral particles infective viral particles

�� PathognomicPathognomic of HPV infection of HPV infection

�� KoilocyteKoilocyte with HPV test neg. is with HPV test neg. is

confusing for ptconfusing for pt

�� UK decided not to report UK decided not to report koilycytekoilycyte in in

cytology report at sentinel sites for HPV cytology report at sentinel sites for HPV

triage with low grade abnormalitytriage with low grade abnormality

HPV reporting HPV reporting

Cytology Cytology vsvs Molecular test Molecular test

Should we be replacing cytology Should we be replacing cytology

with HPV Molecular Test?with HPV Molecular Test?

Dilemma of Cytology negative BUT HPV positive Molecular Dilemma of Cytology negative BUT HPV positive Molecular TestTest

�� Epithelial cells are not producing virus particlesEpithelial cells are not producing virus particles

�� Patient is likely to be in immunologic equilibrium with Patient is likely to be in immunologic equilibrium with the virusthe virus

�� Virus will eventually disappearVirus will eventually disappear

�� Transmission of virus to sexual partner (s) at this stage Transmission of virus to sexual partner (s) at this stage is doubtfulis doubtful

�� Eventual outcome of the infection is not knownEventual outcome of the infection is not known�� Require long term followRequire long term follow--up study to establish longup study to establish long

term FU protocolterm FU protocol

ARTISTIC Trial is extended for further 6 yearsARTISTIC Trial is extended for further 6 years

Modern Modern KoilocyteKoilocyte is is

Moving Moving

ToTo

The Molecular LBC SmearsThe Molecular LBC Smears

with HPV Testwith HPV Test

AND AND

NOT Retiring Yet!NOT Retiring Yet!

The EndThe End

AcknowledgementAcknowledgement

My special thanks to My special thanks to

Dr. Dr. Rana,DrRana,Dr. Denton & Dr. . Denton & Dr. BijalBijal Shah Shah

From UKFrom UK

&&

Mr.Andrew Mr.Andrew EveredEvered

From Wales Training SchoolFrom Wales Training School

ForFor

Producing images and drawingsProducing images and drawings

andand

Writing some of the textsWriting some of the texts

I have also used I have also used HologicHologic CytycCytyc ThinprepThinprep Images, BD Images, BD SurepathSurepath Images andImages and

Images from NHSCSP atlasImages from NHSCSP atlas

For this presentationFor this presentation