Papanicolaou Staining (Pap stain) for Pap Smear / Pap Test

Papanicolaou staining (Pap stain) is a widely used technique for staining cells in cytological and histological preparations. It is named after George Papanicolaou, who developed it in 1942. The Pap stain is also known as the Pap smear, which is the procedure of collecting and staining cells from various body sites for examination under a microscope.

The Pap stain is a polychromatic stain, which means it uses multiple dyes to stain different components of the cells with different colors and intensities. The Pap stain can reveal the morphology, structure, and function of the cells, as well as detect any abnormalities or infections that may be present.

The Pap stain is mainly used for screening cervical cancer, which is one of the most common cancers in women. By examining the cells scraped from the cervix, the Pap stain can identify precancerous or cancerous changes in the cervical epithelium. The Pap stain can also be used to screen for other types of cancers, such as thyroid, liver, and bladder cancers, as well as to diagnose benign tumors, inflammatory conditions, and microbial infections.

The Pap stain is a simple, inexpensive, and effective method for screening and diagnosis of various diseases. However, it is not a definitive test and requires further confirmation by other techniques, such as biopsy or molecular tests. The Pap stain also has some limitations in terms of sensitivity, specificity, and accuracy, depending on the quality of the sample collection, staining procedure, and interpretation.

In this article, we will discuss the objectives, principle, composition, procedure, results, interpretation, applications, and limitations of the Papanicolaou staining technique. We will also provide some examples and images of the Pap stain to illustrate its use and significance.

Papanicolaou staining (Pap stain) is a cytological staining technique that aims to achieve three main objectives for the examination of cell samples :

• Definition of nuclear details: The Pap stain highlights the nuclear features of the cells, which are often abnormal in cancerous or precancerous conditions. The stain uses hematoxylin, a natural dye that binds to the DNA molecules and stains the nuclei blue. This allows the pathologist to assess the size, shape, and chromatin pattern of the nuclei and identify any signs of malignancy or dysplasia .
• Transparency of cytoplasm: The Pap stain also makes the cytoplasm of the cells transparent and visible under the microscope. This is important because the cells in the smear preparations may vary in thickness and overlap with each other. The transparency of the cytoplasm helps to distinguish different cell types and layers, as well as to detect any cytoplasmic inclusions or alterations .
• Differentiation of cells: The Pap stain uses two counterstains, orange G-6 and eosin azure, which are acidic dyes that stain the basic components of the cytoplasm. These counterstains help to differentiate various cell types based on their staining reactions and cytoplasmic characteristics. For example, orange G-6 stains mature and keratinized cells orange, while eosin azure stains metabolically active cells such as columnar cells and intermediate squamous cells blue-green .

By achieving these objectives, the Pap stain enables the pathologist to examine the cell samples in a comprehensive and accurate manner and to diagnose various diseases and infections. The Pap stain is most commonly used for screening cervical cancer in gynecological smears (Pap smears), but it can also be applied to other specimens containing exfoliated or aspirated cells from various body sites .

The principle of Papanicolaou staining is based on the differential staining of cellular components by basic and acidic dyes. Basic dyes have a positive charge and stain acidic components of the cell, such as DNA and RNA, which have a negative charge. Acidic dyes have a negative charge and stain basic components of the cell, such as proteins and cytoplasm, which have a positive charge.

Papanicolaou staining uses five dyes in three solutions as the main reagents:

• Hematoxylin: This is a natural basic dye that stains the nuclei of the cells blue. It has a high affinity for nuclear chromatin, which contains DNA and RNA. Hematoxylin binds to the sulfate groups of DNA and RNA molecules. The most common hematoxylin dyes used in Papanicolaou staining are Harris’ hematoxylin, Gills’ hematoxylin and Hematoxylin S.

• Orange G 6: This is an acidic counterstain that stains the cytoplasm of mature keratinized cells orange. Keratin is a protein that forms the outer layer of the skin and other tissues. Orange G 6 binds to the amino groups of keratin molecules.

• Eosin Azure: This is a combination of three acidic counterstains: eosin Y, light green SF and Bismarck brown Y. Eosin Y stains the cytoplasm of mature squamous cells, nucleoli, red blood cells and cilia pink. Squamous cells are flat cells that line the surface of the cervix and other organs. Nucleoli are structures within the nuclei that produce ribosomes. Red blood cells are cells that carry oxygen in the blood. Cilia are hair-like structures that help move fluids along the respiratory tract. Eosin Y binds to the amino groups of these cellular components. Light green SF stains the cytoplasm of active cells, such as columnar cells, parabasal squamous cells and intermediate squamous cells, blue-green. Columnar cells are tall cells that line the inner surface of the cervix and other organs. Parabasal squamous cells are immature squamous cells that are found near the base of the epithelium. Intermediate squamous cells are transitional squamous cells that are found between the parabasal and superficial layers. Light green SF binds to the carboxyl groups of these cellular components. Bismarck brown Y does not stain anything and is sometimes omitted from the solution.

The Papanicolaou staining procedure involves applying these reagents in a specific order and duration to achieve optimal differentiation and contrast between different cell types and structures. The procedure also involves rinsing, dehydrating and clearing steps to remove excess dye and prepare the smear for mounting and examination under a microscope.

The Papanicolaou staining technique allows for the identification and classification of normal and abnormal cells in a smear preparation. It is especially useful for detecting cervical cancer and its precursors by examining changes in the shape, size, color and arrangement of cervical cells.

The main reagents used in Papanicolaou staining are:

• Hematoxylin: This is a natural dye that stains the cell nuclei blue. The dye attaches to the sulfate groups of DNA because it has a high affinity for nuclear chromatin. The most common hematoxylin dyes used are Harris’ hematoxylin, Gills’ hematoxylin and Hematoxylin S.

• Orange G 6: This 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.

• Eosin Azure: This 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. 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.

The composition of these reagents can vary depending on the manufacturer and the protocol used. However, a typical composition of these reagents is as follows:

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

Other reagents include 95% ethanol, 100% ethanol, tap water, Scott’s tap water, xylene.

The basic procedure for Papanicolaou staining involves the following steps:

1. Fix the smear with 95% ethanol for 15 minutes. This step preserves the cellular morphology and prevents the loss of nuclear details.
2. Rinse in tap water to remove excess ethanol and prepare the smear for staining.
3. Add Harris hematoxylin dye for 1-3 minutes. This dye stains the nuclei blue by binding to the sulfate groups of DNA.
4. Rinse in tap water or Scott`s tap water to remove excess hematoxylin and differentiate the nuclei. Scott`s tap water is a weak alkaline solution that enhances the blue color of the nuclei by removing excess acid from the hematoxylin.
5. Dip the preparation in 95% ethanol for 10 dips to dehydrate the smear and prepare it for counterstaining.
6. Add orange G-6 stain for 1.5 minutes. This stain is an acidic counterstain that stains the cytoplasm of mature keratinized cells orange in varying intensities depending on the degree of keratinization.
7. Dip in 95% ethanol for 10 dips to remove excess orange G-6 stain and dehydrate the smear further.
8. Add eosin azure stain for 2.5 minutes. This stain is 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. Light green SF stains the cytoplasm of active cells such as columnar cells, parabasal squamous cells, and intermediate squamous cells blue-green. Bismarck brown Y stains nothing and is sometimes omitted.
9. Dip in 95% ethanol for 10 dips, 2 changes to remove excess eosin azure stain and dehydrate the smear completely.
10. Add 100% ethanol for 1 minute to further dehydrate the smear and prepare it for clearing.
11. Clear in 2 changes of xylene, 2 minutes each to remove ethanol and make the smear transparent and ready for mounting.
12. Mount with permanent mounting medium such as Canada balsam or synthetic resin and cover with a coverslip.

The Papanicolaou staining technique produces a polychromatic effect, meaning that different components of the cells are stained with different colors and intensities. This helps to differentiate various types of cells and identify any abnormalities in their morphology. The main results and interpretation of the Pap stain are as follows:

• Nuclei: The nuclei of the cells are stained blue by the hematoxylin dye, which binds to the acidic DNA molecules. The intensity of the blue color depends on the amount and distribution of chromatin in the nuclei. Normal nuclei are usually small, round, and uniform in size and shape. Abnormal nuclei may be enlarged, irregular, hyperchromatic (dark blue), or hypochromatic (light blue), indicating malignancy or dysplasia.
• Cytoplasm: The cytoplasm of the cells is stained by the orange G6 and eosin azure dyes, which bind to the basic proteins and lipids in the cytoplasm. The color and intensity of the cytoplasmic stain depend on the type and maturity of the cells. Mature squamous cells have pink cytoplasm due to eosin Y, while immature squamous cells have blue-green cytoplasm due to light green SF. Columnar cells, parabasal cells, and intermediate cells also have blue-green cytoplasm. Keratinized cells have orange-red cytoplasm due to orange G6.
• Other structures: The Pap stain also stains other structures that may be present in the smear, such as red blood cells, bacteria, fungi, parasites, and inflammatory cells. Red blood cells are stained orange-red by eosin Y, while bacteria are stained blue by hematoxylin. Fungi such as Candida are stained red by eosin Y, while parasites such as Trichomonas are stained gray-green by light green SF. Inflammatory cells such as neutrophils, lymphocytes, macrophages, and eosinophils are stained according to their nuclear and cytoplasmic characteristics.

The interpretation of the Pap stain results is based on the Bethesda System (TBS), which is a standardized system for reporting cervical cytology findings. TBS has three main categories: negative for intraepithelial lesion or malignancy (NILM), epithelial cell abnormalities (ECA), and other malignant neoplasms (OMN). Each category has subcategories that describe the type and severity of the abnormality detected. For example, ECA includes atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), atypical glandular cells (AGC), and squamous cell carcinoma (SCC). OMN includes adenocarcinoma, neuroendocrine carcinoma, lymphoma, melanoma, and metastatic tumors.

The Pap stain is a screening test that can detect cervical cancer and precancerous lesions at an early stage. However, it is not a diagnostic test and it has some limitations, such as low sensitivity, false negatives, false positives, and sampling errors . Therefore, any abnormal Pap stain result should be confirmed by further tests such as colposcopy, biopsy, or HPV testing.

Papanicolaou staining has various applications in cytology and histology, where it is used to differentiate and identify cells in smear preparations from different sources. Some of the applications are:

• Used in the Pap smear (or Pap test): This is the most common and important application of Papanicolaou staining, where it is used to screen for cervical cancer and other precancerous or malignant lesions of the cervix, vagina, and vulva. The Pap smear involves collecting cells from the cervix using a spatula or a brush and staining them with Papanicolaou stain. The stained cells are then examined under a microscope for any abnormal changes in their morphology, such as enlarged nuclei, irregular shapes, or altered cytoplasmic colors. The Pap smear can also detect infections by human papillomavirus (HPV), which is a major risk factor for cervical cancer .
• Examination of other body fluids and secretions: Papanicolaou staining can also be used to stain cells from various body fluids and secretions, such as sputum, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspirates, tumor biopsies, etc . These specimens can be used to diagnose or monitor various diseases and conditions, such as lung cancer, bladder cancer, meningitis, tuberculosis, peritonitis, pleurisy, arthritis, infertility, etc .
• Examination of tissue sections: Papanicolaou staining can also be modified and applied to tissue sections from various organs and tissues . This can help to identify and characterize different types of cells and tissues in histopathology. For example, Papanicolaou staining can be used to stain thyroid tissue sections to detect thyroid cancer, or to stain liver tissue sections to examine myeloma cancer cells.

Papanicolaou staining is a widely used and valuable technique for screening and diagnosis of various diseases and infections. However, it also has some limitations that should be considered when interpreting the results. Some of the limitations are:

• It is only a screening test that must be followed up with more specialized diagnostic tests. Papanicolaou staining can detect abnormal cells or microorganisms in a smear, but it cannot confirm the diagnosis or the cause of the abnormality. For example, if a pap smear shows atypical squamous cells of undetermined significance (ASCUS), it does not necessarily mean that the patient has cervical cancer or human papillomavirus (HPV) infection. Further tests such as colposcopy, biopsy, or HPV testing are needed to confirm the diagnosis and guide the treatment.
• It has low sensitivity with limited accuracy. Papanicolaou staining may miss some cases of disease or infection due to various factors such as inadequate sampling, poor fixation, improper staining, or human error. The sensitivity of pap smear for detecting cervical cancer ranges from 30% to 87%, depending on the quality of the sample and the interpretation of the results. The accuracy of pap smear for identifying specific microorganisms such as Candida or Chlamydia is also low, as they may be confused with other cells or artifacts in the smear.
• It is influenced by various physiological and pathological factors. Papanicolaou staining may show different results depending on the stage of the menstrual cycle, hormonal status, pregnancy, menopause, inflammation, infection, or medication use of the patient. These factors may affect the appearance and distribution of the cells in the smear, making them difficult to classify or interpret. For example, estrogen may cause an increase in superficial cells and glycogen content in the cytoplasm, while progesterone may cause an increase in intermediate and parabasal cells and keratinization. Inflammation may cause cellular changes such as nuclear enlargement, hyperchromasia, or multinucleation that may mimic dysplasia or malignancy.
• It requires skilled personnel and quality control. Papanicolaou staining is a complex and subjective technique that requires trained and experienced cytotechnologists and pathologists to perform and interpret the results. The quality of the staining and the interpretation may vary depending on the skill and judgment of the personnel involved. Quality control measures such as regular calibration of equipment, standardization of reagents, internal and external quality assurance programs, and proficiency testing are essential to ensure the reliability and consistency of the results.

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