Papanicolaou Staining (Pap Stain) – Principle, Procedure & Results

Papanicolaou staining (Pap stain) is commonly used in the cytological examination of cervical smears and other cytological specimens. A cervical smear contains exfoliated epithelial cells collected from the cervix. After collection, the cytological material is processed in the laboratory for slide preparation, fixation and staining before microscopic examination. Proper handling and processing of the specimen help in preserving the cellular morphology required for microscopic examination.

What is Papanicolaou Staining (Pap Stain)?

Papanicolaou staining or Pap stain is a polychromatic staining technique used for the examination of cells in cytology. It involves the use of different dyes to stain the nucleus and cytoplasm of a cell. The cell nuclei show clear details, while the cytoplasmic components take up different stains. This staining method is used in both gynecological and nongynecological cytology. Cervical smears, body fluids and fine-needle aspiration specimens are stained by this method.

The method was developed by George N. Papanicolaou and is named after him. Pap is a shortened form of his surname. It is not an acronym.

Pap staining and the Pap test are not the same. Pap staining is the laboratory staining method, whereas the Pap test is used for cervical screening. In a Pap test, cells collected from the cervix are examined for abnormalities. The collected cervical cell specimen is known as a Pap smear. The term Pap smear is also commonly used for the Pap test.

Purpose of Papanicolaou Staining

The following are the purposes of Papanicolaou staining:

• To obtain clear nuclear staining for the examination of nuclear chromatin, size and shape.
• To differentiate the cytoplasmic characteristics of cells, including keratinized and non-keratinized cells.
• To maintain the transparency of the stained cytoplasm so that overlapping cells can be examined under the microscope.
• To identify different types of epithelial cells based on their nuclear and cytoplasmic characteristics.
• It is used to examine cellular abnormalities, including changes in nuclear morphology, chromatin pattern and cytoplasmic appearance.

Principle of Papanicolaou Staining

The principle of Papanicolaou staining is based on the different affinities of nuclear and cytoplasmic components for the staining dyes. Hematoxylin is used to stain the cell nucleus. It is oxidized to hematein, which combines with a mordant, usually an aluminium salt, and binds to the nuclear material.

Hematoxylin can be used by progressive or regressive staining. In progressive staining, the nuclei are stained to the required intensity. In regressive staining, the nuclei are first overstained with hematoxylin, and the excess stain is removed by differentiation using a weak acid solution. Bluing is carried out in a weakly alkaline medium, which changes the reddish nuclear stain to blue or purple.

Orange G-6 is an acidic cytoplasmic counterstain which mainly stains keratinized cells orange. Eosin-azure (EA) contains different dyes used for cytoplasmic staining. Eosin stains the cytoplasm of mature squamous cells pink, while light green stains other cytoplasmic components green or blue-green.

The staining colour depends upon the chemical nature of the dyes, pH of the staining solution and composition of the cells. Different cytoplasmic proteins have different affinities for these dyes. Proper fixation and alcohol-based cytoplasmic staining help in maintaining cellular transparency. The nuclear chromatin is clearly visible, while the cytoplasm remains transparent even in overlapping cells.

Reagents and Materials Required for Papanicolaou Staining

The following are the stains, reagents and materials required for Papanicolaou staining. The composition of the staining solutions may differ according to the formulation used.

Primary Stains and Counterstains

1. Hematoxylin is used for staining the cell nuclei. Harris and Gill’s hematoxylin are commonly used preparations containing an aluminium salt as a mordant.
2. Orange G-6 (OG-6) is an alcoholic counterstain containing Orange G dye. It stains the keratinized cytoplasm orange.
3. Eosin-Azure (EA) is a polychromatic cytoplasmic counterstain. EA-50 and EA-65 are commonly used preparations which differ in the proportions of the dyes.
4. Eosin Y is an acidic dye present in EA solutions. It stains the cytoplasm of mature squamous cells pink.
5. Light Green SF yellowish is used for staining the cytoplasm of metabolically active cells green or blue-green. Some commercial EA preparations contain Fast Green FCF in place of Light Green SF yellowish.
6. Bismarck Brown Y is present in certain traditional EA formulations. It is omitted from modified EA preparations because it has little effect on the staining colour and may cause precipitation.
7. Phosphotungstic acid is added to OG-6 and EA staining solutions to maintain acidic conditions and control dye uptake. Acetic acid is also present in certain preparations for maintaining the required pH and improving cytoplasmic staining.

Fixatives and Other Reagents

1. 95% ethanol is commonly used as a wet fixative for cytological smears.
2. Graded alcohol solutions are required for hydration and dehydration during staining. Absolute ethanol is used for the final dehydration of stained slides.
3. Acid alcohol is used for the differentiation of hematoxylin in the regressive staining method. It is generally not required for progressive hematoxylin staining.
4. Bluing solutions such as Scott’s tap water substitute, dilute ammonium hydroxide or lithium carbonate solution are used for bluing the stained nuclei. Slightly alkaline tap water may also be used.
5. Distilled water is required for the preparation of staining solutions and washing of slides.
6. Xylene is used as a clearing agent before mounting. Validated xylene substitutes may also be used.
7. A permanent mounting medium, such as DPX or a compatible synthetic resin, is required for mounting the stained slides.

Materials and Equipment

1. Clean glass microscope slides for preparing cytological smears.
2. Staining jars or Coplin jars for holding the staining solutions.
3. Staining racks for holding and transferring the slides.
4. Forceps for handling the microscope slides.
5. Reagent bottles and containers for storing the stains, alcohol solutions and other reagents.
6. Glass coverslips for mounting the stained specimens.
7. A light microscope for examining the stained cells.

Preparation and Fixation of Cytology Smears

The preparation and fixation of cytology smears for Papanicolaou staining are given below:

1. The collected cellular material is placed on a clean glass slide and spread gently to prepare a thin, even smear.
2. The smear should contain sufficient cells for microscopic examination. Thick smears contain overlapping cells and clumps which may obscure the nuclear and cytoplasmic details.
3. Very thin smears may contain too few cells for examination. Excessive pressure during spreading can also cause distortion of the cells.
4. The prepared smear is fixed immediately while it is still wet. This is known as wet fixation.
5. For Papanicolaou staining, 95% ethanol is commonly used as a fixative. The slide is immersed in the fixative to preserve the nuclear and cytoplasmic morphology of the cells.
6. Air drying of the smear before fixation should be avoided. Delayed fixation may cause changes in cellular morphology and poor staining of the nucleus and cytoplasm.
7. Other validated alcohol-based fixatives, such as methanol and isopropanol, may also be used. Commercial spray fixatives containing alcohol and a protective coating are another alternative.
8. The duration of fixation depends upon the type of specimen, fixative used and laboratory protocol.
9. In liquid-based cytology, the collected cells are transferred directly into a vial containing a preservative solution instead of preparing a conventional smear. The cells are then processed in the laboratory and placed on a glass slide to form a relatively uniform layer.

Procedure of Papanicolaou Staining

Conventional Papanicolaou Staining Procedure

The following is the conventional Papanicolaou staining procedure using the Sigma-Aldrich HT40 Standard Method (2025). The staining time of hematoxylin depends upon the formulation used.

1. Prepare a thin smear on a clean glass slide and immediately fix it in 95% ethanol for at least 15 minutes.
2. Remove the slide from 95% ethanol and rinse it with tap water.
3. Stain the smear with Harris or Gill’s hematoxylin for 1–3 minutes.
4. Wash the slide with tap water to remove excess hematoxylin.
5. For regressive hematoxylin staining, differentiate the smear in the ready-to-use differentiation solution for 20–60 seconds. This step is not required for progressive staining.
6. Rinse the slide with tap water and transfer it to Scott’s tap water substitute for 10 dips. The working solution is prepared by mixing one part concentrate with nine parts deionized water.
7. Wash the slide with tap water and transfer it to 95% ethanol for 10 dips.
8. Stain the smear with Orange G-6 (OG-6) for 1.5 minutes.
9. Rinse the slide in 95% ethanol for 10 dips.
10. Counterstain with EA-50, EA-65 or modified EA-65 for 2.5 minutes.
11. Wash the slide in two changes of 95% ethanol, 10 dips in each.
12. Transfer the slide to absolute ethanol (100%) for 1 minute.
13. Clear the slide in two changes of xylene or a validated xylene substitute for 2 minutes each.
14. Mount the stained smear with a compatible permanent mounting medium and place a coverslip over it.

Rapid Papanicolaou Staining Procedure

Rapid, economical, acetic acid Papanicolaou (REAP) staining is a modified Pap staining method used to reduce staining time and alcohol consumption. The following procedure uses the REAP method described for cervical smears, with Harris hematoxylin, OG-6 and EA-50.

1. Prepare a thin cytological smear and immediately fix it in 95% ethanol.
2. Transfer the fixed slide to 1% acetic acid for 10 dips.
3. Stain the smear in Harris hematoxylin preheated to 60°C for 10 dips.
4. Wash the slide in tap water for 10 dips.
5. Transfer the slide to 1% acetic acid for 10 dips.
6. Stain the smear with Orange G-6 for 10 dips.
7. Rinse the slide in 1% acetic acid for 10 dips.
8. Counterstain the smear with EA-50 for 10 dips.
9. Wash the slide in 1% acetic acid for 10 dips.
10. Dehydrate the slide in methanol for 10 dips.
11. Clear the slide in xylene for 10 dips.
12. Mount the stained smear with DPX mounting medium and cover it with a coverslip.

In the REAP method, 1% acetic acid is used in place of most intermediate alcohol solutions. Ordinary tap water is used for bluing, and the exposure time of the stains is reduced to 10 dips. The hematoxylin is heated in a temperature-controlled water bath without an open flame.

The ultrafast Papanicolaou staining method is different from REAP staining. It is used for rapid examination of fine-needle aspiration smears and may involve rehydration of air-dried smears with normal saline, followed by alcoholic formalin fixation and different staining reagents.

Results and Interpretation of Papanicolaou Staining

Staining Results

The Papanicolaou-stained smear is examined under a light microscope. The cell nuclei and cytoplasm take up different stains and show different colours.

The following are the common staining results of Papanicolaou staining:

The nuclear chromatin is clearly visible, while the cytoplasm remains transparent. This allows the examination of overlapping cells under the microscope. The staining colour may vary according to the staining formulation, pH, fixation method and composition of the specimen.

Microscopic Interpretation

The stained cells are examined for their nuclear and cytoplasmic characteristics. The nuclear outline, size, chromatin distribution and presence of nucleoli are observed. The size of the nucleus is also compared with the amount of cytoplasm to determine the nuclear-to-cytoplasmic ratio.

Superficial squamous cells usually have a small, dense nucleus with abundant cytoplasm. Intermediate cells have a relatively larger nucleus with finely distributed chromatin. Parabasal cells contain comparatively less cytoplasm and have a higher nuclear-to-cytoplasmic ratio.

Abnormal cells may show enlarged nuclei, irregular nuclear membranes, coarse chromatin and an increased nuclear-to-cytoplasmic ratio. Prominent nucleoli may also be present in certain reactive or abnormal cells. These changes are examined along with the cytoplasmic staining characteristics for the recognition of cytological atypia.

Inflammation, cellular repair and poor fixation may also produce changes in nuclear and cytoplasmic appearance. The staining colour alone cannot be used to identify a particular disease or establish a final clinical diagnosis.

In cervical cytology, the Bethesda System is used for reporting the microscopic findings and epithelial cell abnormalities. These diagnostic categories are different from the staining results of Papanicolaou staining.

Precautions and Quality Control of Papanicolaou Staining

The following are the precautions and quality control measures for Papanicolaou staining:

• Fix the smear immediately after preparation. Air drying may distort the cells and affect the nuclear details.
• Prepare a thin, even smear on a clean glass slide. Thick smears may stain poorly due to overlapping cells.
• Use 95% ethanol for wet fixation. Diluted or contaminated alcohol may cause poor fixation and staining.
• Replace the alcohol when its concentration is no longer suitable.
• Maintain the required hematoxylin staining time. Insufficient staining gives pale nuclei, while excessive staining makes the chromatin difficult to observe.
• Avoid excessive differentiation with acid alcohol in regressive staining. Too much differentiation removes the nuclear stain, while insufficient differentiation leaves the nuclei dark.
• Use a suitable alkaline solution for bluing. Incomplete bluing may leave the nuclei reddish or purple.
• Maintain the required staining time for OG-6 and EA counterstains. Weak or exhausted stains may give pale cytoplasmic staining.
• Replace exhausted counterstains or adjust the staining time according to the laboratory protocol.
• Avoid prolonged treatment with alcohol after cytoplasmic staining. It may remove the cytoplasmic dyes.
• Filter the staining solutions regularly to remove dye precipitates and cellular debris.
• Keep the staining solutions covered to prevent alcohol evaporation and moisture entry, which may change their concentration and cause precipitation.
• Use clean staining jars, racks and forceps to prevent contamination between slides. Replace contaminated reagents.
• Dehydrate the smear completely with fresh absolute alcohol. Residual water or contaminated xylene may cause cloudiness and poor transparency.
• Use a compatible permanent mounting medium and place the coverslip without air bubbles. Avoid drying of the smear before mounting and the use of excess mounting medium.
• Examine a stained control slide regularly for clear nuclear chromatin, cytoplasmic colour and transparency. Record staining problems and correct or replace the reagents before further staining.
• Wear gloves, a laboratory coat and safety glasses while handling the reagents. Alcohols and xylene are flammable, and xylene vapours are harmful.
• Handle clearing solvents in a suitable fume hood. Keep the chemicals away from open flames and dispose of waste according to laboratory safety procedures.

Applications of Papanicolaou Staining

The following are the applications of Papanicolaou staining:

• It is used in the Pap test for screening cervical precancerous changes and cancer.
• To examine abnormal squamous and glandular cells in cervical smears.
• Used in vaginal cytology for studying the maturation of epithelial cells.
• It is used in urine cytology to detect abnormal urothelial cells.
• Sputum, bronchial washings and brushings are stained to examine cells of the respiratory tract.
• It is used for cytological examination of pleural, peritoneal and pericardial fluids.
• Fine-needle aspiration smears from the thyroid, breast, lymph nodes and other tissues are stained by this method.
• Used for examining epithelial cells collected from other mucosal surfaces by brushing.
• It is also used to examine inflammatory and reactive cellular changes in cytological specimens.

Limitations of Papanicolaou Staining

The following are the limitations of Papanicolaou staining:

• Papanicolaou staining requires immediate fixation of the smear. Air drying may distort the cells and affect the nuclear details.
• Thick smears contain overlapping cells, making microscopic examination difficult.
• Blood, mucus and inflammatory cells may obscure the cellular details.
• Smears containing insufficient cells may not be suitable for examination.
• Poor staining may occur due to incorrect staining time, dye concentration or pH of the solutions.
• Prolonged treatment with alcohol may remove the cytoplasmic stains and reduce their colour intensity.
• Abnormal cells may be missed if they are not present in the collected specimen.
• Reactive and inflammatory changes may resemble abnormal or malignant cells.
• Some microorganisms can be observed, but their identification may require additional staining or laboratory tests.
• It cannot determine tissue invasion because the tissue structure is not preserved in cytological smears. A biopsy may be required for confirmation.
• Conventional Papanicolaou staining requires several staining and washing steps, making it time-consuming.
• Proper preparation, staining and interpretation require trained laboratory personnel.

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