A Review of Pap Smear Techniques: Laboratory Procedure

Pap smear, a cervical screening technique.

Introduction

Pap smear shortened from Papanicolau test, is a cytological screening test used to check for cervical cancer and its precursors.

The Pap smear process involves collecting cells from a patient’s cervix using a speculum for better visualization. Then a spatula, brush, or swab is used to collect cells from the zone of the ‘junctional mucosa’ (the gradual transition from squamous to columnar/glandular epithelial cells) of the cervix.

Pap smear (collecting, fixation, and staining of cervical cells) was first developed in the 1940s, by Dr. G. N. It has since become an important diagnostic tool for identifying abnormal cells that may indicate dysplasia, carcinoma in situ, or invasive cervical cancer.

 

Overview of the Cervix

The cervix is a part of the female reproductive organ that connects the vagina to the uterus.

It consists of two parts;

The Ectocervix is the part connecting the vagina and with squamous epithelial cells.

The Endocervix lines the inner part of the cervix and consists of columnar epithelial cells.

The junction where the two epithelial cells meet is called the squamous columnar junction (SCJ) or junctional mucosa.

Why is this important in pap smear testing?

This information is important because pap smear is collected from the SCJ. It is a clinically and pathologically significant landmark in the cervix. In other words, any change in this zone is a pointer towards something.

Different physiological and cytological changes occur in the cervix, at varying stages of a woman’s life.  This is also noteworthy as this helps in the preparatory phase for sample collection

 

Techniques of Pap smear

 

1. Preparation for pap smear test:

• The ideal time for scheduling your pap smear test is 10-20 days after your menstrual period. This is to prevent blood from interfering with the result.
• Avoid intercourse, douching, or using vaginal products 3 days before the scheduled day for pap smear, as this may change the normal constitutions of the cells. And we don’t want anything that will interfere with the result.

 

2. Sample collection

Pap smear is collected from the cervix by a healthcare professional, with the aid of a speculum to visualize the patient’s cervix. A spatula or brush is inserted through the speculum into the cervical os and rotated through 360 degrees; gently scarping cells from the transformation zone where the outer squamous cells meet the inner glandular cells.

 

3. Fixation

• Conventional Pap Smears: After collection, the cells are directly smeared onto a microscope slide and is immediately fixed using a spray fixative or alcohol-based solution to preserve the cellular morphology.
• Liquid-Based Cytology (LBC): The sample is transferred into a preservative fluid. The cells are suspended in the liquid preservative and transported to the laboratory, where they are processed to produce a thin layer of cells on a slide.

This method has become more common due to improved cell preservation and the ability to remove non-cellular debris, making the slides clearer.

 

4. Staining

Papanicolaou Staining:

This multichromatic staining technique involves several steps to highlight different cellular components.

• Hematoxylin: Stains the nuclei a blue to purple color, which is critical for assessing nuclear abnormalities.
• Orange G (OG): Stains keratinized cells orange, which is helpful for identifying certain types of epithelial cells.
• EA (Eosin Azure): Stains the cytoplasm of cells in various shades, helping to distinguish different types of epithelial cells (e.g., mature squamous cells vs. glandular cells).

           Staining procedure:

• Fix the smears while wet in a cytological fixative for at least 30mins
• Rinse in descending grades of alcohol(80,70,50%) and water for 10seconds each
• Stain in Harris haematoxylin (without acetic acid) for 2mins
• Rinse in water
• Differentiate in acid alcohol for few seconds
• Wash and blue in tap water for 5mins
• Transfer in ascending grades of alcohol (70,95%) for few minutes each
• Stain in OG6 for 2minuites
• Rinse in 2 changes of 95% alcohol
• Stain in EA50 for 3-4 mins
• Rinse in 2 changes of 95% alcohol
• Dehydrate in absolute alcohol
• Clear in xylene and mount in neutral synthetic medium

Pap smear staining process is also automated.

Note: There are other stains used in Pap smear staining.

 

5. Result

Nuclei ——————————————-Blue

Acidophilic (superficial cells)————-Red-Orange

Basophilic (intermediate and parabasal cells)—Blue/Green

        Candida albican————————–Red – Pink

        Trichomonas vaginalis——————Grey Green

 

6. Slide screening

• Manual screening: A cytotechnologist or cytopathologist screens the slides under the microscope, perusing for abnormal cells.
• Automated Screening systems: Some laboratories utilize automated systems like ThinPrep imaging System or Hologic’s imager, to pre-scan the slides and flag areas of interest for further manual review.

 

7. Result Interpretation and Report

Bethesda System for Reporting Cervical Cytology is widely used as it provides a standardized framework for reporting Pap smear results. It includes categories such as:

• Negative for intraepithelial lesion or malignancy.
• Atypical squamous cells of undetermined significance (ASC-US).
• Low-grade squamous intraepithelial lesion (LSIL).
• High-grade squamous intraepithelial lesion (HSIL).
• Squamous cell carcinoma.

 

Reference

• Ochei, J. and Kolhatkar, A. (2008) Medical Laboratory Science, Theory and Practices. Tata McGraw-Hill, New York, 311-347
• Textbook of Pathology. Author, Harsh Mohan. Edition, 6. Publisher, Jaypee Brothers, 2010. ISBN, 8171797830, 9788184487022. Length, 949 pages

 

We will explore more on Pap smear and the underlying structures surrounding it in subsequent posts.