PAP test methods

Cervical Cancer : Pap smear
 George N Papanicolaou introduced cervical cytology in
clinical practice in 1940
 In 1945, PAP smear was endorsed by American cancer
society as an effective method for prevention of cervical
cancer
 Many countries now have National cervical screening
programs

Indian scenario
 Commonest cancer in women in India
 Major cause of deaths in women due to cancer
 Usually diagnosed at advanced stage
 No National program
 Uniformly low incidence of cervical screening in India
(6% in rich & 4% in poor)

Histological Types 30
 Squamous Cell Carcinoma : 80-95%
 Adenocarcinoma : 5-20%
 Other : Clear cell, sarcomas

Transformation zone
 Cervix develops from 2 embryonic sites
* from Mullerian duct - lined by columnar epithelium
* from urogenital plate - lined by stratified
squamous epithelium
 Point at which columnar and squamous epithelium
meet is called as original squamo-columnar
junction

Transformation zone
 Under influence of estrogen, original SCJ moves
onto the portio.
 Exposure of delicate columnar cells to vaginal
environment leads to squamous metaplasia.
 Transformation zone -
- Area of squamous metaplasia
- Area between original and new SCJ

Transformation Zone -TZ
 Exposure of TZ to carcinogens begins the process of
intraepithelial neoplasia
 While exact role of carcinogens in this process remains poorly
understood, it is clear that HPV and cigarette smoking can
cause dysplasia at the TZ
 95% of cervical cancers develop in TZ
 Important to take sample from TZ

Transformation Zone
 Transformation zone may not be viewed during
routine speculum examination

Why cervical screening is a
feasible and useful strategy?
 Relative accessibility of cervix to take the smear
 Long natural history of cervical carcinogenesis
 Relative conservative treatment for premalignant lesions
 Cost effectiveness3

PAP Smear
 PAP smear sampling of cervix involves scraping of
cervical surface and a portion of non visualised
cervical canal using various sampling devices

Significance of Pap smear
 Detect precancerous & invasive cancer cervix cases in
early stages
 Positive screeners can be selected for selective tests and
management
 With treatment, progression of disease is halted. Thus
morbidity associated with advanced cancer decreases
 Mortality reduces by 20-60 %.
 Helps us to study natural history of disease.

Cervical Cancer : Pap smear
 Early detection of pre-malignant lesions by Pap
smears prevent at least 70% of potential cervical
cancers.

Of the 30% who actually
develop cervical cancer:
 8% elude cytological detection
- imperfections in cytological technology
- biologic behavior of malignant lesions
 22% represent women who develop cervical cancer because of
failure to regularly seek Pap smears => women whose cancers
could have been prevented with early detection and treatment.

How to take a Pap Smear ?
 Proper technique is very important
 More problems are due to improper sampling than
screening
 Not to be collected during menses
 Avoid vaginal contraceptives, vaginal medications for at
least 48 hrs before taking smear
 Abstinence for 24 hrs
 Postpartum smear should be taken only after 6 - 8 weeks of
delivery

 Patient in dorsal position
 Good illumination is necessary
 Cusco’s speculum is inserted to visualise & fix
the cervix
 Inspection of cervix done & findings are noted
 Ayres spatula is inserted first. It is placed at
cervical os so that longer end goes into cervical
canal and smaller end rests on ectocervix

 Spatula is rotated through 360 degrees
maintaining contact with ectocervix
 Do not use too much force [bleeding /pain]
 Do not use too less force [inadequate sample]
 Sample is smeared evenly on the slide and fixed
immediately
 Both sides of spatula are to be smeared

 Endocervical sample is collected using an
endocervical brush
 Insert the cytobrush into canal, so that last bristles
of brush are visible
 Rotate the brush through 180 degrees. [more
rotations increase the chance of bleeding]
 Sample is rolled on the slide and fixed.

Fixation of smear
 Fixation is done immediately with
fixative like 95% alcohol or cytofix
spray to avoid air drying
 Spray should be kept at 10 inches, to
avoid destruction of cells by
propellent in the spray
 Smear should monolayer for proper
penetration of cell surface by fixative

 Slide should be labeled properly with patients name,
identification no. and details
 Detailed history and clinical examination findings are to be
mentioned
 Patient details and clinical findings are to be maintained in a
register
 Advice is given regarding further follow up and treatment

Systems for cervical cytology reporting
 George N Papanicolaou (1954)
5 classifications based on certainty of finding malignant cells
 Descriptive system – WHO - (1968)
based on morphologic criteria – included mild, moderate,
severe dysplasia and Ca In Situ
 Richart – CIN –based on histologic diagnosis

Systems for cervical cytology reporting
 Bethesda system – TBS (1988)
National cancer institute revised in 1991 and 2001
 Adequacy of smear must be determined before reporting
Smear is adequate when
- Patient identification
- adequate clinical history

Bethesda system
 Interpretable cellular cytology
 not obscured by inflammation, debris, blood, drying
 not scanty smear
 Adequate sampling from transformation zone
 presence of at least 2 clusters of well preserved
endocervical cells or metaplastic cells

Bethesda system
Results :
 Within normal limits ( WNL )
Benign cellular changes - this term was removed and
group was included in WNL in 2001
 Reactive or Reparative changes – seen with atrophy,
inflammation, surgery, radiation, IUCD, tampoons
 Infections – trichomoniasis, fungal, bacterial, HSV.

Bethesda system - results
 Epithelial cells abnormalities
 Squamous cells
• ASCUS
• ASCUS-H - suggestive of high grade lesion
• LSIL - changes associated with HPV, atypical
changes, mild dysplasia/ CIN1
• HSIL – moderate to severe dysplasia / CIN2, 3
and Ca In Situ
• HSIL – where invasion cannot be ruled out
• Squamous cell carcinoma

Bethesda system
Results :
 Glandular cells – AGUS (Endocervical, endometrial)
Adenocarcinoma
(endocervical, endometrial, extrauterine)
 Other malignant neoplasms

Normal cervix-cytology
 Squamous cells
 Exfoliated indivisual cells
 Navicular in shape with abundant cytoplasm and small,
dark, round /oval, pyknotic nuclei
 Glandular cells
 Many times seen in clumps - linear or honeycombed
pattern.
 Slightly larger and basal nuclei

Cervical cytology - Inflammation
 Interpretation difficult due to inflammatory
background
 Lot of neutrophils and blood can obscure
cellular details

High grade lesions
High grade squamous
lesion
High grade glandular
lesion

Abnormal Pap smear- HPV
 Peripherial condensation of cytoplasm -
wire looping effect
 Koilocyte

PAP Descriptive CIN Bethesda
Class-1 negative negative WNL
Class 2
Inflammatory,
squamous, koilocytic
atypia
Reactive, reparatative
changes, ASCUS,
LSIL(HPV)
Class 3
Mild dysplasia
Moderate dysplasia
Severe dysplasia
CIN1
CIN2
CIN3
LSIL(HPV)
HSIL
HSIL
Class 4 Ca In Situ CIN3 HSIL
Class 5 Invasive Invasive Invasive

 Single test will not detect cervical abnormality but with 3
negative tests there is less than 1% chance of cervical
abnormality
 Conventional cytology has specificity of 98% and
sensitivity of 51%.
PAP smear

PAP Smears - Limitations
 Low sensitivity 51%
 False negative rates are due to faulty sampling, improper
fixation or interpretation problems
 Large group population & high risk group screening not
possible
 No consensus regarding testing

Pap smear as screening method
 New guidelines
 Target group - All women aged 18-70 yrs who have ever had sex
 Timing of Initial Screening -
Initial screening at age of 21 years or within 3 years of sexual activity
ACOG Guidelines-(Aug2003), American Cancer Society (Nov 2002) and
U.S. Preventative Services Task Force (Jan 2003)

Pap smear - guidelines
Screening interval - yearly till the age of 30 then 3 yearly
 When to End Screening
- After 70 yrs
- Post Hysterectomy
- done for benign lesions
- previous 3 normal PAP reports
- confirmed complete removal of cervical epithelium

Pap smear - guidelines
 In high risk group after treatment for CIN
every 3 monthly for 2 years
 every 6 monthly for 3yrs
 Yearly thereafter
 Women who had hysterectomy for CIN, it is necessary to do
vault smears
 In women who received vaccination against HPV, it is
necessary to continue screening

Liquid Based Cytology
 To improve results of PAP newer techniques like liquid
based cytology are recommended
 Cells are obtained with a broom, then the head is broken
off in to a vial containing preservative fluid
 In the laboratory the sample is spun to remove obscuring
material
 It gives clearer image, no cell clumps
 It will assist in future automated reading

 Several slides can be prepared from one smear
 Chlamydia, HPV testing can be done at later date
 Reduces the incidence of inadequate and repeat smears
Liquid Based Cytology

Cancer Cervix IS PREVENTABLE ,
IF Detected EARLY!!!!!!!!!
Thank You

PAP test methods

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PAP test methods