PAP SMEAR

PAP SMEAR INTRODUCTION

Cancer of the cervix (cervical cancer) is the fourth most common cause of cancer-
related death among women worldwide. The best way to detect cervical cancer is by
having regular Papanicolaou tests, or Pap smears. (Pap is a shortened version of the
name of the doctor who developed the screening test.) A Pap smear is a microscopic
examination of cells taken from the uterine cervix.

A Pap smear can detect certain viral infections such as human papillomavirus (HPV),
which is known to cause cervical cancer. Early treatment of precancerous changes
detected on the Pap smear can stop cervical cancer before it fully develops. A woman
may have cervical cancer and not know it because she may not have any symptoms.

The incidence of cancer and deaths from cervical cancer has significantly declined over
the years because of prevention, screening, and early detection by the Pap smear. Most
abnormal Pap smear results indicate the early stages of disease and need reasonable
observation by a doctor.

Risks factors for cancer of the cervix include conditions that increase the likelihood of
being infected with HPV as well as other factors including the following:

 Multiple sexual partners (or sexual partners who have had multiple partners)
 Starting sexual intercourse at an early age
 Weakened immune system
 Previous cancer of the lower genital tract
 Smoking

New recommendations were published in March, 2012 by the U.S. Preventive Service
Task Force with agreement by the American Cancer Society (details are described
further in this article). Briefly stated, cervical cancer screening is now recommended
every 3 years starting at age 21. Screening may be carried out every 5 years for women
over age 30 if a Pap smear and HPV test are performed.

No upper age limit for screening exists because the incidence of cancer of the cervix
increases with age at a time when women may be less likely to get a Pap smear.
Diagnosis of most of these cancers is in women older than 50 years. Even after
menopause, a woman should continue to have regular Pap smears. Women over age
65 or older who have had three or more normal Pap tests in a row and no abnormal Pap
test results in the last 10 years may choose to stop having Pap tests.

If a woman has had her uterus removed, she should still have yearly screening if there
is a history of advanced precancerous changes seen on Pap smear or other lower
genital tract cancer.

PAP SMEAR RISKS

The Pap smear procedure is not complicated or painful. The only risk is not detecting
cervical cancer in time to treat and cure it

PAP SMEAR PREPARATION

The best time to have a Pap smear is when the woman is not menstruating. For two
days before the test, avoid the following because these might interpretation of the test
more difficult:

 Intercourse
 Douches
 Vaginal medications (except as directed by your doctor)
 Vaginal contraceptives such as birth control foams, creams, or jellies

DURING THE PROCEDURE

A Pap smear is usually part of a pelvic exam and accompanied by a breast exam
performed by the health care professional. It should only take about one minute to
perform a Pap smear during this overall exam.

 The woman will lie on the examination table (see Multimedia File 1) on her back
with her knees up and bent and her feet in stirrups (rests). While she is lying on
an examination table, her health care professional will use a small metal or
plastic instrument called a speculum to open the vagina so that the walls of the
vagina and cervix can be seen clearly.

 A sample of mucus and cells will be obtained from the cervix (see Multimedia
File 2) (the part of the uterus that extends into the vagina) and endocervix (the
 opening of the cervix) using a wooden scraper or a small cervical brush or
broom.

 Formerly, a sample of cells was evenly applied to a glass slide and sprayed with
a fixative. This sample was sent to the lab for close and careful examination
under a microscope. Currently almost all providers are using a new kind of Pap
smear called a ThinPrep test, the sample is rinsed into a vial and sent to a lab
for slide preparation and examination.

 A cytologist (a specialist trained to look at the cells and interpret a Pap smear)
reviews both types of tests.

 Some discomfort during the test may occur. Most women feel nothing at all or
feel pressure. Staying relaxed will help stop any discomfort. The woman should
breathe slowly and concentrate on relaxing her stomach and legs.

 A Pap smear should not be painful. If a woman has pain during the test, she
should tell her doctor.

AFTER THE PROCEDURE

The health care professional will send a letter with test results. If there is a problem, the
woman's health care professional may contact her. For peace of mind, she can also call
the medical office to get the results. Before leaving after the exam, she can ask how
long it takes the office to receive the lab report.

A negative or normal test findingmeans that the cervix looks healthy. All the cells are
of a healthy size and shape.

A positive or abnormal test findingmeans that something unusual is in the sample.

The test found cells of a different size and shape.

An abnormal Pap smear result does not always indicate cancer. Cells sometimes
appear abnormal but are not cancerous. The woman will have to return to the doctor for
follow-up care.

 An infection of the cervix may cause an abnormal test result. Yeast,

trichomonas, chlamydial, or gonorrheal infection can cause the cervical cells to
appear inflamed. After the infection is treated, the Pap smear result usually
returns to normal.
  If the Pap smear result is positive because of an infection, the underlying cause
should be treated. The test should then be repeated in 2-3 months, because
cancer of the cervix can be hidden by an infection. A check-up with a doctor is
necessary.

Most laboratories in the United States use a standard set of terms called the Bethesda
System to report, or interpret, test results. Under the Bethesda System, Pap smear
samples that have no cell abnormalities are reported as "negative for
intraepithelial lesion or malignancy" (meaning the woman does not have cancer).

Samples with cell abnormalities fall into the following categories (as outlined by the
National Cancer Institute):

 ASC (atypical squamous cells): Squamous cells are the thin, flat cells that
form the surface of the cervix. The Bethesda System divides this category into
the following two groups:

o ASC-US (atypical squamous cells of undetermined significance): The

squamous cells do not appear completely normal, but doctors are
uncertain what the cell changes mean. Sometimes the changes are
related to HPV infection. ACS-US isconsidered a mild abnormality.

o ASC-H (atypical squamous cells cannot exclude a high-gradesquamous

intraepithelial lesion): The cells do not appear normal, but doctors are
uncertain what the cell changes mean. ASC-H may be at higher risk of
being precancerous.

 AGC (atypical glandular cells): Glandular cells are mucus-producing cells

found in the endocervical canal (opening in the center of the cervix) or in the
lining of the uterus. The glandular cells do not appear normal, but doctors are
uncertain what the cell changes mean.

 AIS (endocervical adenocarcinoma in situ): Precancerous cells found in the

glandular tissue.

 LSIL (low-grade squamous intraepithelial lesion): Low-grade means there

are early changes in the size and shape of cells. The word lesion refers to an
area of abnormal tissue. Intraepithelial refers to the layer of cells that forms the
surface of the cervix. LSILs are considered mild abnormalities caused by HPV
infection.

 HSIL (high-grade squamous intraepithelial lesion): High-grade means that

there are more marked changes in the size and shape of the abnormal
 (precancerous) cells, meaning the cells look very different from normal cells.
HSILs are more severe abnormalities and have a higher likelihood of
progressing to invasive cancer.

PAP SMEAR FOLLOW-UP

If a woman's Pap smear result is normal, she will continue routine screening.

If the Pap smear result is abnormal, the doctor will recommend repeat testing or more
frequent follow-up, depending upon the exact type of abnormality and whether any
infection is present. The doctor may choose to do a procedure known as acolposcopy.

 In this test, the doctor looks at the cervix through an instrument called a
colposcope (a lighted microscope) to look for an explanation for the abnormality
in the Pap smear finding. It is performed in the office in a manner similar to the
Pap smear, but the doctor uses a special viewing instrument that magnifies the
appearance of the surface of the cervix to examine the area for abnormalities.

 The exam is not painful and has no adverse effects. It is possible to perform this
exam during pregnancy.

 If there are abnormal cells on the cervix, the doctor will perform a biopsy (take a
sample of the tissue to view under a microscope).

 In a biopsy, the doctor will take a small sample of the tissue of the woman's
cervix to see if cancer cells are present. A biopsy is the only way to determine if
she has precancer, true cancer, or neither.

 Several types of biopsies are performed under different types of anesthesia.

 To treat precancer tissue or a very early cancer, the doctor may remove the
abnormal tissue entirely during certain types of biopsy methods.

PAPANICOLAOU (PAP) STAINING

Papanicolaou stain (also Papanicolaou’s stain or PAP stain) is the most important stain
utilized in the practice of Cytopathology. It is a polychromatic stain containing multiple
dyes to differentially stain various components of the cells. This technique was
developed by George Papanicolaou, the father of Cytopathology. This method is used to
differentiate cells in the smear preparation of various gynecological specimens (pap
smears), materials containing exfoliative cells and material from fine needle aspiration.

OBJECTIVES OF PAPANICOLAOU STAIN

Papanicolaou described three chief objectives for staining of cytological smears:

 Definition of nuclear details : Because of the widespread muclear

abnormalities of cancer cells and their diagnostic significance, good staining of
the nucleus is of primary importance.
 Transparency of cytoplasm : This is of particular importance because of the
varying thickness and the frequent overlapping of cells.
 Differentiation of cells : Differences in the staining reaction such as that
between acidophilic and basophilic cells help greatly in the identification of
certain cell types found in smears.

PRINCIPLE OF PAPANICOLAOU STAIN

Papanicolaou stain includes both acidic and basic dyes. Acidic dye stains the basic
components of the cell and basic dye stain the acidic components of the cell. The
polychromatic stain involves five dyes in three solutions.
1. Hematoxylin : Natural dye hematoxylin is the nuclear stain which stains cell
nuclei blue. It has affinity for chromatin, attaching to sulphate groups on the
D.N.A. molecule. Harris’ hematoxylin is the commonest cytologically although
Gills’ hematoxylin and Hematoxylin S can be used.
2. Orange Green 6 : This is the first acidic counterstain (cytoplasmic stain) which
stains matured and keratinized cells. The target structures are staine d orange in
different intensities.
 3. Eosin Azure : This is the second counterstain which is a polychrome mixture of
eosin Y, light green SF and Bismarck brown.
Eosin Y gives a pink colour to cytoplasm of mature squamous cells, nucleoli,
cilia and red blood cells. Staining solutions commonly used in cytology are EA 31
and EA 50, while EA 65
Light green SF stains blue to cytoplasm of metabolically active cells like
parabasal squamous cells, intermediate squamous cells and columnar cells.
Bismarck brown Y stains nothing and sometimes it is often omitted.

COMPOSITION AND PREPARATION OF REAGENTS

1. Harris’ hematoxylin :
Hematoxylin = 5g
Ethanol = 50ml
Potassium alum = 100g
Distilled water (50°C) = 1000ml
Mercuric oxide = 2-5g
Glacial acetic acid = 40ml
2. Orange G 6 :
Orange G (10% aqueous) = 50ml
Alcohol = 950ml
Phosphotungstic acid = 0-15g
3. EA 50 :
0.04 M light green SF = 10ml
0.3M eosin Y = 20ml
Phosphotungstic acid = 2g
Alcohol = 750ml
Methanol = 250ml
Glacial acetic acid = 20ml
Filter all stains before use.
PROCEDURE OF PAPANICOLAOU STAINING

Both progressive and regressive nuclear staining techniques can be used in

Papanicolaou stain. Before staining, Wet fixation immediately with Cytology spray
fixative 96% ethanol for minimum 30 min is required.

Procedure of Progressive Papanicolaou Staining Method

In the progressive method, the nucleus is stained with hematoxylin to a intensity

desired. The intensity of the nuclear staining is controlled by the immersion of the slide
into a blueing agent. Most commonly used blueing agent is Sott’s tap water (pH 8.02).

Step Reagent Time

1. 95% Alcohol (Fixation) 15-30 minutes

2. 80% Alcohol 2 minutes

3. 60% Alcohol 2 minutes

4. Distilled Water 5 dips

5. Distilled Water 5 dips

6. Hematoxylin stain 3 minutes

7. Distilled Water 3 minutes

8. 60% Alcohol 2 minutes

9. 80% Alcohol 2 minutes

10. 95% Alcohol 2 minutes

11. Orange G Stain 3 minutes

12. 95% ALcohol 2 minutes

13. 95% Alcohol 2 minutes

14. Eosin Azure Stain 3 minutes

15. 95% Alcohol 2 minutes

16. 95% Alcohol 2 minutes

17. 95% Alcohol 2 minutes

18. 95% Alcohol 2 minutes

19. Absolute Alcohol 2 minutes

20. Absolute Alcohol 2 minutes

21. Absolute Alcohol 2 minutes

22. Absolute Alcohol+Xylene (1:1) 2 minutes

23. Xylene 2 minutes

24. Xylene 2 minutes

25. Xylene Till clear

26. Mount in D.P.X

Procedure of Regressive Papanicolaou Staining Method

When using the regressive staining method, the nucleus is deliberately over-stained
with a non-acidified haematoxylin. The excess stain is removed with dilute hydrochloric
acid solution (acid water). The decolourising process is then stopped by immersing the
slide in running tap water. Timing is crucial in the regressive method as de-staining may
lead to a hyperchromatic nucleus becoming hypochromatic.

Step Reagent Time

1. 90% Alcohol (Fixation) 15-30 minutes

2. 80% Alcohol 2 minutes

3. 60% Alcohol 2 minutes

4. Distilled Water 5 dips

5. Distilled Water 5 dips

6. Hematoxylin stain 3 minutes

7. Distilled Water 10 seconds

8. 1% Acid Alcohol 10 seconds (1 dip)

9. Distilled Water 10 seconds

10. Scott’s Tap Water 2-3 minutes

11. Running Tap Water 2 minutes

12. 60% Alcohol 2 minutes

13. 80% Alcohol 2 minutes

14. 95% Alcohol 2 minutes

15. Orange G Stain 3 minutes

16. 95% ALcohol 2 minutes

17. 95% Alcohol 2 minutes

18. Eosin Azure Stain 3 minutes

19. 95% Alcohol 2 minutes

20. 95% Alcohol 2 minutes

21. 95% Alcohol 2 minutes

22. 95% Alcohol 2 minutes

23. Absolute Alcohol 2 minutes

24. Absolute Alcohol 2 minutes

25. Absolute Alcohol 2 minutes

26. Absolute Alcohol+Xylene (1:1) 2 minutes

27. Xylene 2 minutes

28. Xylene 2 minutes

29. Xylene Till clear

30. Mount in D.P.X

RESULTS AND INTERPRETATION OF PAPANICOLAOU STAINING

 Nuclei : Blue
 Acidophilic cells : Red
 Basophilic cells : Blue Green
 Erythrocytes : Orange-red
 Keratin : Orange-red
 Superficial cells : Pink
 Intermediate and Parabasal Cells : Blue Green
 Eosinophil : Orange Red
 Candida : Red
 Trichomonas : Grey green

FROZEN TISSUE STAINING

The following is a general procedure guide for preparation and staining of acetone-
fixed frozen tissues using a purified, unconjugated primary antibody, biotinylated
secondary antibody and streptavidin-horseradish peroxidase (Sav-HRP) and DAB
detection system. Because each

antigen differs in terms of requirement for fixation, amplification step, etc., it Is not
possible to write an inclusive protocol that will work for all antigens. The user must
determine optimal conditions for each antigen of interest. Many protocols for staining
individual antigens, as well as useful tips and troubleshooting guides for
immunohistochemistry, can be found at the IHCWorld web site

Procedure guide for immunohistochemical staining on frozen tissue sections:

Prepare frozen tissue sections (steps 1-8):

1. Place a freshly dissected tissue block (<5 mm thick) on to a pre-labeled tissue

base mold.

2. Cover the entire tissue block with cryo-embedding media (e.g. OCT).

3. Slowly place the base mold containing the tissue block into liquid nitrogen till the
entire tissue block is submerged into liquid nitrogen to ensure tissue is frozen
completely.

4. Store the frozen tissue block at -80°C until ready for sectioning.

5. Transfer the frozen tissue block to a cryotome cryostat (e.g. -20°C) prior to
sectioning and allow the temperature of the frozen tissue block to equilibrate to the
temperature of the cryotome cryostat.

6. Section the frozen tissue block into a desired thickness (typically 5-10 µm) using
the cryotome.

7. Place the tissue sections onto glass slides suitable fo immunohistochemistry (e.g.
Superfrost).
8. Dry the tissue sections overnight at room temperature. Sections can be stored in
a sealed slide box at -80°C for later use. Immunostain frozen tissue sections (steps
9-28):

9. Fix the tissue sections with a suitable fixative. One of the commonly used fixation
methods for frozen tissue sections is to immerse the slides in pre-cooled acetone (-
20°C) for 10 min.

10. Pour off the fixative and allow acetone to evaporate from the tissue sections for >
20min at room temperature.

11. Rinse the slides in 300 ml of 10 mM phosphate buffered saline (PBS) at a neutral
Ph for 2 changes, 5 min each.

12. Incubate the slides in 0.3% H2O2 solution in PBS at room temperature for 10
min to block endogenous peroxidase activity.

13. Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

14. (optional) Add 100 µl blocking buffer (e.g. 10% fetal bovine serum in PBS) onto
the sections of the slides and incubate in a humidified chamber at room temperature
for 1h.

15. Drain off the blocking buffer from the slides.

16. Apply 100 µl an appropriately diluted primary antibody (in antibody dilution buffer,

e.g. 0.5% bovine serum albumin in PBS) to the sections on the slides and incubate
in a humidified chamber for 1 h at room temperature or overnight at 4°C.

17. Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

18. Apply 100 µl an appropriately diluted biotinylated secondary antibody (using the
antibody dilution buffer) to the sections on the slides and incubate in a humidified
chamber at room temperature for 30 min.

19. Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

20. Add 100 µl pre-diluted Sav-HRP conjugates (using the antibody dilution buffer) to
the sections on the slides and incubate in a humidified chamber at room temperature
for 30 min (keep protected from light).

21. Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

22. Apply 100 µl DAB substrate solution (freshly made just before use: 0.05% DAB -
0.015% H2O2 in PBS) to the sections on the slides to reveal the color of the
antibody staining. Allow the color development for < 5 min until the desired color
intensity is reached.

(Caution: DAB is a suspected carcinogen. Handle with care. Wear gloves, lab coat
and eye protection.)

23. Wash slides in 300 ml PBS for 2 changes 5 min each.

24. (optional) Counterstain slides by immersing sides in Hematoxylin (e.g. Gill’s

Hematoxylin)for 1-2 min.

25. Rinse the slides in running tap water for > 15 min.

26. Dehydrate the tissue slides through 4 changes of alcohol (95%, 95%, 100% and
100%), 5min each.

27. Clear the tissue slides in 3 changes of xylene and coverslip using mounting
solution

(e.g. Permount). The mounted slides can be stored at room temperature

permanently.

28. Observe the color of the antibody staining in the tissue sections under
microscopy.
MUSCLE - PHOSPHOTUNGSTIC ACID-HEMATOXYLIN, MALLORY'S (PTAH)

PURPOSE: Used for diagnosing muscle cross-striations and fibrin.

PRINCIPLE: The amount of phosphotungstic acid in the staining solution is far

greater than the amount of hematein and it is believed that tungsten binds all
available components blue, while the phosphotungstic acid is thought to stain the
red-brown components. This stain has been referred to as a polychrome stain
because one solution gives two major colors. The components colored red-brown
will lose this color with water or prolonged alcohol washes, and dehydration of the
section following staining therefore must be rapid.

CONTROL: Use skeletal or cardiac muscle, tissue containing fibrin for fibrin staining,
and cerebral cortex for glial fibers.

FIXATIVE: Zenker's fixative preferred or formalin fixed.

TECHNIQUE: Cut paraffin sections 4m for muscle, 6m for glial fibers.

EQUIPMENT: Coplin jars, microwave oven. Rinse all glassware in DI water.

REAGENTS:

Lugol's:

See Stock Solutions

5% Hypo:

See Stock Solutions

PTAH Stain

0.25% Potassium Permanganate:

Potassium permanganate 0.25 gm

Distilled water 100.0 ml

Mix fresh, discard after use.

CAUTION: Strong oxidant.

5% Oxalic Acid Solution:

Oxalic acid 5.0 gm

Distilled water 100.0 ml

Mix well, label with initial and

date. Solution is stable for 1 year.

CAUTION: avoid contact and inhalation

Zenker’s Fluid:

Distilled water 500.0 ml

Mercuric chloride 25.0 gm

Potassium dichromate 12.5 gm

Sodium sulfate 5.0 gm

Mix well, label with date and initial. Solution is stable indefinitly.

Working Solution:

Right before use, to 50 ml of

Zenker’s add 5 ml glacial aceticacid. Discard after use.

CAUTION: Corrosive poison, discard in labeled glass container.

SAFETY: Wear nitrile gloves and lab coat, wear a particle mask or work

under the hood. Keep all hot uncapped solutions under the hood. Avoid

contact and inhalation. Zenker’s contains; Mercuric chloride: Severe skin and eye
irritant; target organ effects on reproductive, urogenital, respiratory, gastrointestinal
and fetal systems via ingestion and inhalation routes. Potassium dichromate: toxic
by inhalation of dust and by ingestion; target organ effects on reproductive and fetal
systems. Solid form is corrosive to eyes, skin and mucous membranes. Iodine:
dermal sensitizer, irritant to eyes, skin and respiratory system. Sodium thiosulfate:
Toxic on ingestion. Can irritate the stomach. Irriant to skin, eyes and respiratory
tract. Potassium permanganate: Skin and eye irritant, ingestion will lead tosevere
gastrointestinal distress. Strong oxidant. Oxalic acid: can cause severe burns of the
eyes, skin or mucous membranes. Toxic by inhalation and ingestion. Target organ
effects on kidneys and cardiovascular system, repeated exposure can cause
dermatitis. Corrosive.

PROCEDURE:

1. Deparaffinize and hydrate to distilled water.

2. *Zenker's fixative, microwave Hi power, 45 seconds, allow to stand

for 5 minutes.

3. Wash with tap water.

4. Lugol's iodine for 5 minutes.

5. Wash in tap water.

6. 5% hypo for 1-2 minutes.(for glial fibers, bleach out iodine in 95%

alcohol in place of hypo.)

7. Wash in tap water 10 minutes.

8. Oxidize in 0.25% potassium permanganate, 5 minutes.

9. Wash in tap water.

10. Bleach in 5% oxalic acid until white.

11. Wash in tap water, rinse in distilled water.

12. PTAH stain, overnight, room temperature.

13. Dehydrate rapidly through two changes each of 95%, 100% alcohol, and

two changes of xylene, coverslip.

RESULTS:

Cross-striations, fibrin, glial fibers - blue

Nuclei - blue

Collagen red-brown

Elastic fibers purplish

 3

ASSIGNMENT IN HISTOPATHOLOGY-
LABORATORY

BERSAMINA, HANNA GRACE D.

BSMT 4-1

201310805

SUBMITTED TO: MA’AM ISOLDE QUITAN