INTRODUCTION ABOUT CYTOLOGY AND ITS TECHNIQUES

1. PRESENTED BY
D. JASMINE PRIYA, B.Sc., DCA., M.Sc., PGDCLT.
DR. NGP ARTS AND SCIENCE COLLEGE
COIMBATORE

2. CYTOPATHOLOGY – HISTORY
 Born in Kyme on the Island of Euboea in Greece on May 13th, 1883, Dr George N. Papanicolaou, referred
to as the ‘Father of Exfoliative Cytology’, was undoubtedly an outstanding doctor, scientist and
humanitarian in modern medical history.
 He studied Medicine at the University of Athens, Greece, where he received his MD in 1904 and he
completed his PhD at the University of Munich in Germany in 1910.
 On October 19th, 1913, he moved to New York accompanied by his wife, Mache, known as Mary
Papanicolaou, and one year later, in September 1914, he joined the Cornell University Medical College's
Department of Anatomy in New York, where he performed his scientific work for the rest of his life.
 In 1915, he published his first article in ‘Science’ on sex determination in guinea pigs
 After 1923, Dr Papanicolaou clarified the correlation of the vaginal smear cytology with the ovarian
cycle in pregnant and non-pregnant women and discovered that women with cervical cancer
exhibited ‘abnormal cells, with enlarged, deformed or hyperchromatic nuclei’.

3.  In January 1928, he presented his findings, entitled as ‘New Cancer Diagnosis’, at the Third Race
Betterment Conference in Battle Creek, Michigan, where he introduced his low-cost, easily
performed screening test for early detection of cancerous and precancerous cells.
 However, this potential medical breakthrough was initially met with skepticism and resistance from
the scientific community at that time, and his technique was initially considered an unnecessary
addition to the existing diagnostic methods for cervical cancer.
 Almost 10 years later, in 1939, at the encouragement of the gynaecologist Dr Herbert Traut, Dr
Papanicolaou continued his work in this field and on March 11th, 1941, Papanicolaou and Traut
published their findings in their paper entitled ‘The diagnostic value of vaginal smears in
carcinoma of the uterus’ .
 At this time, the Pap smear-test technique won acceptance and soon became widely accepted as a
routine screening methodology, worldwide.

4. GEORGE N. PAPANICOLOAU

5. What is cytology?
 Cytology is an Greek word. It means
 Cyto – cells
 Pathology –study about disease
 Cytology (also known as cytopathology) involves examining cells from bodily tissues or fluids to
determine a diagnosis.
 A certain kind of scientist called a pathologist will look at the cells in the tissue sample under a
microscope and look for characteristics or abnormalities in the cells.
 Since cytology only examines cells, which are so tiny, pathologists only need a very small sample of
tissue to do a cytology test.
 Healthcare providers use cytology in many different areas of medicine, but cytology tests are most
commonly used to screen for or diagnose cancer.
 Cytopathology is a branch of pathology that studies and diagnoses diseases on the cellular level. The
discipline was founded by George Nicolas Papanicolaou in 1928.

6.  Cytopathologic tests are sometimes called smear tests because the samples may be smeared across a
glass microscope slide for subsequent staining and microscopic examination.
 However, cytology samples may be prepared in other ways, including cytocentrifugation.
 Different types of smear tests may also be used for cancer diagnosis. In this sense, it is termed
a cytologic smear.
 Cytopathology is generally used on samples of free cells or tissue fragments, in contrast
to histopathology, which studies whole tissues.
 Cytopathology is frequently, less precisely, called "cytology", which means "the study of cells“.
Who performs a cytology test?
 Depending on the type of cytology test, many types of healthcare providers could collect the sample of
cells. For example, a gynecologist may take a sample from your cervix for a Pap smear cytology test.
 The healthcare provider then sends the sample to a laboratory for testing.
 A scientist known as a pathologist or cytopathologist looks at the cells from the tissue sample under a
microscope and determines a diagnosis, if applicable.

7. Healthcare providers can use cytology tests for almost all areas of your body. Some
common types of cytology tests include:
• Gynecologic cytology.
• Urinary cytology.
• Breast cytology.
• Thyroid cytology.
• Lymph node cytology.
• Respiratory cytology.
• Eye cytology.
• Ear cytology.

8. SAMPLE COLLECTION
METHODS
INTERVENTION
CYTOLOGY
EXFOLIATIVE
CYTOLOGY

9. 1. What is exfoliative cytology?
Exfoliative cytology is a branch of cytology in which the cells that a pathologist examines are either
“shed” by your body naturally or are manually scraped or brushed (exfoliated) from the surface of your
tissue.
Examples of exfoliative cytology that involve manual tissue brushing or scraping include:
 Gynecological samples: A Pap smear, which involves brushing off cells from your cervix using a
swab, is the most well-known type of exfoliative cytology.
 Gastrointestinal tract samples: Your healthcare provider can brush off cells from the lining of your
gastrointestinal tract (your stomach and intestines) during an endoscopy procedure for cytology
testing.
 Skin or mucus samples: Your healthcare provider can scrape off cells from your skin or mucous
membranes, such as the inside of your nose or mouth, for cytology testing.

10. BRUSHING METHOD SCRAPPING METHOD

11. CYTO BRUSH CYTOLOGY SPATULA

12. Examples of exfoliative cytology that involve collecting tissues or
fluids that your body naturally sheds include:
 Respiratory samples: Your provider can collect fluids such as
spit and mucus (also called phlegm or sputum) that you cough
up for a respiratory cytology test.
 Urinary samples: Your provider can collect a urine (pee)
sample from you to use for a cytology test.
 Discharge or secretion samples: If you experience abnormal
bodily discharge, such as from your eye, vagina or nipple, your
healthcare provider may collect a sample of the discharge for a
cytology test.

13. 2. What is intervention cytology?
 Intervention cytology is a branch of cytology in which your healthcare provider has to “intervene” with your
body to get a sample of cells to test, meaning they have to pierce your skin in some way to get a sample of
cells.
 The most common type of intervention cytology is fine-needle aspiration (FNA).
 A healthcare provider will inject a thin needle into the area that they need to sample and draw out fluid.
 A pathologist then examines the cells in the fluid under a microscope.
 Some areas of your body that a healthcare provider may perform a fine-needle aspiration include:
• Fluid-filled lumps (cysts) under your skin.
• Solid lumps (nodules or masses) under your skin.
• Your lymph nodes.
• Your pericardial fluid, which is the fluid in the sac around
your heart.
• Your pleural fluid, which is in the space between your lung
and the inside of your chest wall.

14.  Fine-needle aspiration, or fine-needle aspiration cytology (FNAC), involves use of a needle attached to
a syringe to collect cells from lesions or masses in various body organs by microcoring, often with the
application of negative pressure (suction) to increase yield.
 FNAC can be performed under palpation guidance (i.e., the clinician can feel the lesion) on a mass in
superficial regions like the neck, thyroid or breast;
 FNAC may be assisted by ultrasound or CAT scan for sampling of deep-seated lesions within the body
that cannot be localized via palpation.
 FNAC is widely used in many countries, but success rate is dependent on the skill of the practitioner.
 If performed by a pathologist alone, or as team with pathologist-cytotechnologist, the success rate of
proper diagnosis is higher than when performed by a non-pathologist.
 This may be due to the pathologist's ability to immediately evaluate specimens under a microscope and
immediately repeat the procedure if sampling was inadequate.

15. FINE NEEDLE CYTOLOGY GUN WITH
SHYRINGE

17. FNAC – PROCEDURE

18. How does a cytology test work?
Each cytology test is slightly different depending on what kind of cells are being tested and if
the sample is tissue or fluid.
In general, there are four steps to a cytology test including:
1. Collecting the sample cells.
2. Processing the sample cells.
3. Examining the sample cells.
4. Sharing the results
1. Collecting the sample cells
Your healthcare provider collects the sample of cells from your body that they need a pathologist to examine.
Some of the ways a provider can collect cytology test samples include:
• Brushing or scraping tissue from the surface of a part of your body.
• Collecting fluid or discharge samples from your body, such as a pee sample.
• Using fine-needle aspiration to draw a fluid sample from an area in your body.

19. 2. Processing the sample cells
 For some types of cytology tests that involve tissue samples, the healthcare provider who took the sample
smears or spreads it on glass microscope slides. These slides are known as smears. They then send the
smears to a pathology laboratory.
 If the cytology test involves bodily fluid, the healthcare provider most likely won’t be able to use smears
since the sample is too diluted (there are only a few cells in the fluid). They’ll most likely send the sample
to a pathology lab in a small container.
 Once a cytology sample arrives at the laboratory, a pathologist or lab technician dips the smears in certain
stains (colored dyes) depending on what kind of sample it is. The stains help make the cells easier to see
and examine under a microscope.
 If the cytology sample is a fluid, a pathologist or lab technician may use a machine called a centrifuge to
separate the cells they want to examine from the fluid. A centrifuge separates certain cells from fluid by
spinning the sample very quickly. The pathologist then puts the cells on smears and may stain them.

20. 3. Examining the sample cells
 After a pathologist or lab technician processes and stains the cytology samples, they examine
the cells under a microscope, looking for abnormal cells.
 If they find abnormal cells, they mark them on the slides with a special pen.
 A pathologist then makes a diagnosis based on the cells and puts together a report.
4. Sharing the results
 After they put together a report, the pathologist will send it to your healthcare provider.
 Your provider will go over the results with you and determine the next steps.

21. Pap Smear
 A Pap smear, also called a Pap test, is an exam a doctor uses to test for cervical cancer in women.
 It can also reveal changes in your cervical cells that may turn into cancer later.
 A pap smear is done to look for changes in cervical cells before they turn into cancer.
 If you have cancer, finding it early on gives you the best chance of fighting it. If you don’t, finding cell
changes early can help prevent you from getting cancer.
 Women ages 21-65 should have a Pap smear on a regular basis.
 How often you do depends on your overall health and whether or not you’ve had an abnormal Pap smear
in the past.
How Often Should I Have a Pap Smear?
 You should have the test every 3 years from ages 21 to 65.
 You may choose to combine your Pap testing with being tested for the human papillomavirus (HPV)
starting at age 30.
 If you do so, then you can be tested every 5 years instead. HPV is the most common sexually transmitted
infection (STI), and it’s linked to cervical cancer.

22. If you have certain health concerns, your doctor may recommend you have a Pap more often.
Some of these include:
 Cervical cancer or a Pap test that revealed precancerous cells
 HIV infection
 A weakened immune system due to an organ transplant, chemotherapy, or chronic
corticosteroid use
 Having been exposed to diethylstilbestrol (DES) before birth
Talk to your doctor if you have questions or concerns. They’ll let you know for sure.

23. Pap Smear Preparation
 You shouldn’t have a Pap smear during your period.
 Heavy bleeding can affect the accuracy of the test. If your test ends up being scheduled for that time of
month, ask your doctor if you can reschedule.
 or the most accurate Pap smear, doctors recommend taking the following steps, starting 48 hours before
your test.
• Don’t have sex or use lubricants.
• Don’t use sprays or powders near the vagina.
• Don’t insert anything into the vagina, including
tampons, medications, creams, and suppositories.
• Don’t rinse the vagina with water, vinegar, or other fluid
(douche).

24. Pap Smear Procedure
 The test is done in your doctor’s office or clinic.
 It takes about 10 to 20 minutes.
 You’ll lie on a table with your feet placed firmly in stirrups.
 You’ll spread your legs, and your doctor will insert a metal or plastic tool (speculum) into
your vagina.
 They’ll open it so that it widens the vaginal walls.
 This allows them to see your cervix.
 Your doctor will use a swab to take a sample of cells from your cervix.
 They’ll place them into a liquid substance in a small jar, and send them to a lab for review.
 The Pap test doesn’t hurt, but you may feel a little pinch or a bit of pressure.

25. PAP STAINING
 Papanicolaou stain is also known as the pap stain and the procedure of the stain is known as a pap smear.
 It is a polychromatic stain that uses multiple dyes to differentially stain various components of the cells.
 It is a histological and cytopathological staining technique used to differentiate cells in a smear
preparation.
 It is the most common screening method for cervical cancer.
 Several specimens can be used to prepare the pap smear depending on the screening infection, including
sputum, urine, cerebrospinal fluid, abdominal fluid, tumor biopsies, synovial fluid, fine needle aspirates,
pleural fluids.
 The technique was developed by George Papanicolaou in 1942.

26. OBJECTIVE
1. 1. To define the cell nuclear to aid in the identification of nuclear abnormalities of cancer cells.
2. To stain the cytoplasm and make it transparent for visualization
3. To differentiate and identify certain cell types such as acidophils and basophils.
PRINCIPLE
The stain uses both basic and acidic dyes such that the basic dye stains acidic components of the cell while the
acidic dyes stain the basic components of the cells. This is based on the ionic charges of the components of the
cell with the principle of attraction and repulsion of the ions and the dyes. Five dyes are used in three solutions
as the main reagents used in the stain.
1. Hematoxylin: This is a natural dye that stains the cell nuclear blue. The dye attaches to the sulfate groups
of DNA because it has a high affinity for nuclear chromatins. The most common hematoxylin dyes used
are Harris’ hematoxylin, Gills’ H is the commonest cytologically although Gills’ hematoxylin and
Hematoxylin S.
2. Orange Green 6: It is an acidic counterstain that stains the cytoplasm of mature keratinized cells. The
components of the target stain orange in varying intensities of the dye.

27. 3. Eosin Azure: It is the second counterstain, a combination of eosin Y, light green SF, and Bismarck
brown. Eosin Y stains the cytoplasm of mature squamous cells, nucleoli, Red blood cells, and cilia pink. The
eosin dyes commonly used are EA 31 and EA 50, while EA 65. Light green SF stains the cytoplasm of active
cells such as columnar cells, parabasal squamous cells, and intermediate squamous cells, blue. Bismarck brown
Y stains nothing and sometimes it is often omitted.
Harris’ hematoxylin
• Hematoxylin = 2.5g
• Ethanol = 25ml
• Potassium alum = 50g
• Distilled water (50°C) = 500ml
• Mercuric oxide = 1-3g
• Glacial acetic acid = 20ml
Orange G 6
• Orange G (10% aqueous) = 25ml
• Alcohol = 475ml
• Phosphotungstic acid = 0-8g
EA 50
• 0.04 M light green SF = 5ml
• 0.3M eosin Y = 10ml
• Phosphotungstic acid = 1g
• Alcohol = 365ml
• Methanol = 125ml
• Glacial acetic acid = 10ml

28. PROCEDURE
1. Fix the smear with 95% Ethanol 15 minutes
2. Rinse in tap water
3. Add the Harris Hematoxylin dye for 1-3 minutes
4. Rinse in tap water or Scott’s tap water
5. Dip the preparation in 95% Ethanol 10 dips
6. Add orange G-6 stain for 1.5 minutes.
7. Dip in 95% Ethanol 10 dips
8. Add Eosin dye; EA-50, or Modified EA-50, or EA-65 stain for 2.5 minutes.
9. Dip in 95% ethanol 10 dips, 2 changes
10. Add 100% Ethanol for 1 minute
11. Clear in 2 changes of xylene, 2 minutes each
12. Mount with permanent mounting medium

29. RESULT
Staining dyes will stain different components of the cell with different colors
and intensities as follows:
• Nuclei: Blue
• Acidophilic cells: Red
• Basophilic cells: Blue Green
• Erythrocytes: Orange-red to dark pink
• Keratin: Orange-red
• Superficial cells: Pink
• Intermediate and Parabasal Cells: Blue-Green
• Eosinophil: Orange Red
• Metaplastic cells: May contain both blue/green and pink
• Candida: Red
• Trichomonas: Grey-green

31. Applications of Papanicolaou Staining (Pap stain)
• Used in the Pap smear (or Pap test).
• Screening for cervical cancer.
• Examination of myeloma cancer cells of the liver.
• Screening for thyroid cancer.
• Screening for cell carcinomas.
• Examination and characterization of benign tumors.
• Identification of Candida species.
• Identification of Chlamydia trachomatis.