PAP SMEAR SCREENING METHOD

DR WAN ROHANI WAN TAIB

DBB40103
Slide Handling
Handles slides by edges only.
Slides should be transported either in slide tray or slide
card holders.
 APPROACH TO EXAMINING A SMEAR
Advisable to adopt a systematic approach.
Cytologist/Lab Staff should:
1- Check identifying information.
Ensure that the specimen is properly labelled and submitted along with the
specific requisition form.
 Match all slides with the requisition form.

 Check names and verify mismatches, if any, and report it to the referring hospital
/ doctor.

 Verify patient’s history including Last Menstrual period, Last Child Birth along with
previous Cytology / Histopathology reports, if any.

 It is mandatory to specify the site from where the specimen has been collected in order
to avoid confusion between a non-gynecological specimen and a cervical smear.

 The number of slides received from each site should be mentioned in the
requisition form.
 Nature and method of sample collection are to be mentioned in the requisition form.
(Cytobrush / Spatula / Swab / for gynaecalogical smears and plain/guided FNAC
for aspiration smears)

 Check whether the fixation is proper. (Type of fixation: alcohol /spray fixative /
prefixed / air dried)

 Separate registers may be maintained for gynaecological, non gynaecological and

sputum (if not computerized.)
 Enter patients name, age, sex, address, brief clinical details, and name of
referring hospital / doctor in the register.

 A unique sequential accession number that will be preceded by the last two digits of
the current year should identify each specimen.

 Mention date and time of receipt of specimen.

 Address to which report is to be despatched should be mentioned in the

requisition form
Match all slides with the requisition form
APPROACH TO EXAMINING A SMEAR
2 . Place slide on the microscope stage with label to the
left.
 APPROACH TO EXAMINING A SMEAR
3. Screen the slides.
 Starting at non label end.
- Magnifications
a) Magnification X 4.
- View adequacy specimen.
-Looking at the overall distribution of the cell
-Marking abnormal cells to be referred
APPROACH TO EXAMINING A SMEAR
b) Magnification X 10.
 Looking at the distribution and morphology of cells
eg: parabasal cells / intermediate / endocervical.
c) Magnification X 40.
 Viewing morphology cytoplasm / nucleus with the
nucleus of the more obvious, such as gross / fine /
pyknotic / vesicular.
 Confirmation for any infections or cells abnormalities
APPROACH TO EXAMINING A SMEAR
4.Screening method.
 Screen in linear track.
 Must screen to edge of slide and also through all edges
of slide again at the end of screening
 Must screen all surfaces of the slide.
 Ensure that all material on the slide is evaluated.
APPROACH TO EXAMINING A SMEAR
2 method for screening.
a) vertical method.

LABEL
APPROACH TO EXAMINING A SMEAR
b) Horizontal method.

LABEL
 Dotted slides

Dotted all unusual finding with ink dot with

X4
Slides are marked with ink dots to identify
cell of interest
Dotted cell should appear to the right of the
dot.
The potential problems in cytology
during screening
• Smear too thick/thin
• Obscuring blood/inflammatory cell
• Abnormal cell too few
• Misinterpretation-false positive/false
negative
• Contaminants & Artefacts
PAPANICOLAOU STAINING
PROCEDURE
 TYPE OF SPECIMEN

 Gynaecological cytology
 Non-gynaecological cytology
(Sputum,Body fluids)
 Smear from Fine Needle Aspiration
cytology

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PAPANICOLAOU STAINING
Polychrome staining
Pap staining was devised for-
 Optimal visualization of cancer cells exfoliated whole
from epithelial surfaces.
 Designed to display many variation of cellular
morphology showing degrees of cellular maturity and
metabolic activity.
 Type of Staining

Manual OR Automation
PAP STAIN
4 main steps Papanicolaou staining procedure:
 Fixation.(95 % ethanol): min 15 min prior to staining. If longer fix in
days or weeks- not affect the morphology of cells. For storage in
fixatives, use capped container in refrigerator.
 Nuclear Staining.
 Cytoplasmic Staining.
 Clearing.
 The Principle of PAP stain
Heamatoxylin: regressive stain, oxidized by mercuric
oxide and form hematin (basic dye)
Basic dye (hematin) will react with acid dye (nucleus)
will differentiate colors for nucleus and cytoplasm.
Differentiated with alcohol acid.
Bluing with Scott water, ammonium water, lithium
carbonate.
Counterstain with Orange G 6 and EA 50.
 Quality control of reagents

Harris Haematoxylin SHOULD BE FILTERED EVERY

DAY before staining process
Sputum is used as CONTROL in PAP stain.
All staining solution SHOULD BE CLOSED if not in used.
Pap Stain(nuclear staining)
 Haematoxylin is used to stain cell nuclei
2 ways to stain nuclei:
a) Regressive method:
Overstained with unacidified hematoxylin ,
then removes the excess stain with diluted
hydrochloric acid (1% acid alcohol).
This de-colorizing acid must be removed by bath
running tap water.
Pap stain(nuclear staining)
b) Progressive method:
This method stains for desired intensity initially with
hematoxylin to which glacial acetic acid has been
added.
The blueing reagent(eg ; Scott solution….) sets the
nuclear dye. Raise pH and turn red color to blue.
Pap stain (nuclear staining)
All formula hematoxylin having a pH of less than 3.0.
Stain at this point is red color.
To change it from red to blue it is necessary to raise
pH to alkaline or between 8.0 -8.5.
By rinsing with tap water or blueing solution
eg-Scott solution ,lithium carbonate.
Pap Stain (cytoplasmic staining)
The purpose of the many colors in the counter stains is
to display the many variations of cellular morphology
showing degrees of differentiation and metabolic
activity.
a) Orange G (OG 6)-
 First cytoplasmic stain is monochromatic
 Intensely colors keratin a brilliant orange.
 Pap Stain(Cytoplasmic staining)
b) EA (EA-50)
 Second cytoplasmic stain is polychrome mixture
composed
 Eosin – Stains cytoplasm of mature squamous
cells(Superficial cells), nucleoli, RBC and cillia.
Light green – Stains cytoplasm of squamous cells
(Parabasal cells,
Intermediate cells), columnar/glandular
cells (Endometrial cells and Endocervical cells)
 Bismarck brown does not add characteristic color to the
cytoplasm. It stains acid mucins to yellow color. It also
stains mast cell granules brown.
PAP STAIN( Clearing)
This final step in the staining procedure result in
cellular transparency.
The solution used will transmit light rays from the
microscope illuminations in the same manner as cells
itself.
Xylene is the most commonly used clearing solution
 PAP
STAIN( Mounting)
1. To preserve the cells for a longer storage
2. To protect the cells from air drying and shrinkage
of cells
3. To avoid oxidation process and fading of colour
Staining procedure: Regressive
1. 95% ethyl alcohol 10 dips
2. 70% ethyl alcohol 10 dips Hydration
3. 50% ethyl alcohol 10 dips
(To prepare for aqueous stain)
4. Rinse in water 1 mins
5. Harris hematoxylin 10 min (overstain)
( Nuclei stain)
6. Running tap water 10 dips 3 mins
( to remove excess hematoxylin)
7. 1.0% acid alcohol 2 dips 
( to decolorize hematoxylin)

8. Running tap water 10 min –(to stop action of acid

alcohol).

9. 50% ethyl alcohol 10 dips

10. 70% ethyl alcohol 10 dips
dehydration
11. 95% ethyl alcohol 15 dips
( to prepare for counterstaining)

12.OG-6 (orange G6) 4 min -

( Cytoplasmic monochromatic stain )
13. 95% ethyl alcohol 5 dips.
(remove excess OG 6)
14. Eosin A 50 – 5 mins
(cytoplasmic polychromatic stain and smear background)
15. 95% alcohol – 5 dips.
16. 95% alcohol – 5 dips Remove excess EA 50 and dehydration
17. Absolute alcohol – 5 dips
18. Absolute aclcohol – 5 dips
19. Xylene - 5 dips
20. Xylene – 5 dips Clearing
21. Depex -mounting
Precautious action in pre and post staining
Reagents
Alcohols for hydration
Alcohol acid - differentiation
Haematoxylin –nuclear staining
OG 6 and EA 50- cytoplasmic and
background staining
Consideration
Enough quantity to avoid uneven
colour distribution and according to the
sequential of gradient to avoid
shrinkage of cells
Dip evenly to ensure the process of
differentiation between positive and
negative stain
Should be filtered to avoid scum or
artefact
Enough quantity to ensure the
coverage of smear staining
Reagents/Kriteria Consideration
Time Should be accurate and optimum for a
good quality of staining
Bluing agent Optimum time to enhance the
haematoxylin effect on nucleus
Air drying The smear should be dried before
mounting to avoid air bubbles that can
damage the cell
Main advantages
Definition of nuclear detail
Cytoplasmic transparency
Cell differentiation
accomplished by
Proper fixation.
Good nuclear stain
Alcoholic counter stain.
Proper clearing
Polychromatic counterstains

Nucleus : blue
cytoplasm : light green, blue-green,
blue or pink
Factors affecting the PAP staining procedure
Type of fixatives: eg Carnoy’s solution, 95% ethanol, ether alcohol, 100%
methanol, 80% propanol, isopropanol
Type of counter stain
Duration of time in each steps
pH of the solution
Temperature of water
Expiry date of the staining solution
Scum or foreign substances in the staining solution: need to be
filtered before use
Type of staining (regressive or progressive)
Quality of smear (thin/thick, air-dried/well fixed): eg if not well
fixed- create artifacts on smear
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Designed by TheTemplateMart.com
Designed by TheTemplateMart.com
Designed by TheTemplateMart.com