Documentation

Introduction

EA 50 reagent is a light green, alcohol solution of two acid dyes: Eosin Y and Light Green SF mixed in Phosphotungstic acid (PTA). It is used in Papanicolaou staining procedure for routine cytoplasmic staining to provide good color contrast and reproducible results that aid in the classification and identification of exfoliative cells. The polychromatic EA solution stains the unstained cellular components, such as nucleoli, squamous cells, cilia, and erythrocytes. The test samples for Papanicolaou staining could be gynecological or non-gynecological including sputum, urine, and cytological samples. EA 50 reacts with the cytoplasm of the less mature squamous cell (exocervical), glandular cell (endocervical), and the basal, paranasal, and intermediate cells.

The Papanicolaou test is a staining procedure used to detect malignant cells in a sample of superficial cells that line the inner wall of the uterine cervix for early cancer diagnosis. The Pap smear is used for cheap and rapid examination of the gynecological and non-gynecological samples.

Properties

Clarity

Clear, may have fine particles.

Appearance

Green-colored solution with a reddish-purple tinge.

Chemical composition

• Denatured alcohol: 100.0ml
• Eosin Y Certified: 0.23gm
• Phosphotungstic acid: 0.2gm
• Bismark Brown Certified: 0.05gm
• Fast green FCF Certified: 0.08gm

The formula of the staining solution can be adjusted and standardized as per the performance parameters.

Objectives of cytological smears staining

Cell differentiation

The differentiation of cells is vital for the proper identification of the cell types found in the cytological smears.

Nuclear details

Since there are many nuclear abnormalities in cancerous cells, the staining of the nucleus is important for an accurate diagnosis.

Cytoplasm transparency

Transparency of the cytoplasm is of particular importance because of the frequent overlapping of cells and their varying thickness.

Principle

The Papanicolaou stain is a mixture of acidic and basic dyes. The acidic part stains the basic components of the cell and the basic stains the acidic components of the cell. The hematoxylin solution contains the nuclear stain which stains the cell nuclei blue. It has an affinity for chromatin particularly the sulfate groups on the DNA molecule. The Orange green 6 is the acidic counterstain which stains the matured and keratinized cells. The Eosin Azure (EA 36, EA 50, and EA 65) is a polychrome mixture of eosin Y, light green SF, and Bismarck brown. Eosin Y gives pinkish shade to the cytoplasm of mature squamous cells, nucleoli, cilia, and red blood cells. Bismarck brown Y stains nothing and is often omitted in cytological staining.

Protocol

Fixation

Fix the sample smears in 95% alcohol for 5-15 minutes.

Staining

1. Rinse the smears in 70% alcohol, 50% alcohol and then in distilled water.
2. Stain in the hematoxylin dye for ten minutes.
3. Rinse again in distilled water.
4. Then rinse in a 0.5% aqueous solution of HCL for 3 to 4 times.
5. Rinse thoroughly in distilled water.
6. Leave the smear in lithium carbonate solution for about a minute and rinse it in distilled water.
7. Rinse again in distilled water and then in increasing alcohol solution concentrations: 50%, 70%, 80%, and 95%.
8. Stain with OG 6 solution for a minute.
9. Then rinse 5-10 times in two jars of 95% alcohol.
10. Stain in EA 50 for 2 minutes.
11. Rinse in two jars of 95% alcohol.
12. Then rinse in absolute alcohol, a mixture of xylene & absolute alcohol and then again in xylene.
13. Mount the smear on the microscope and observe.

Note: The quality of the staining depends on the fixative, age of the staining solution, thickness of the sample, temperature and pH of the water used, rinsing, and air drying.

Results

Following results are obtained with EA 50 staining;

• Keratinized cells: yellow
• Nucleus: crisp blue to black
• Glycogen: yellow
• Cytoplasm: green
• Erythrocytes, superficial squamous epithelial cell, nucleoli, cilia: pink-red

Applications

The EA 50 stain is used in the Papanicolaou staining technique that displays a characteristic range of coloration to exfoliative cells of cervical, vaginal, prostatic and other body secretions facilitating the examination of nuclei and other cytoplasmic components.

Evaluation of bladder neoplasms (Murphy, 1990)

The pathological examination of urinary specimens is vital for the detection and monitoring of the bladder neoplasms. Urinary cytology is one of the diagnostic techniques used for the early diagnosis of urinary neoplasms. The Papanicolaou stain mainly EA 50 is a powerful dye to decipher the cytological details of the urinary sample. The differential diagnosis of urothelial neoplasia versus the reactive/reparative changes secondary to urinary stones could be accomplished. The Papanicolaou staining has rendered new avenues in urinary neoplasia detection, staging, and treatment monitoring.

Strengths and limitations

Eosin Azure 50 is a stable and convenient dye that reacts exclusively with the target cells aiding in their identification and accurate diagnosis. It enables the visualization of nuclear details, keratinized cells, and cytoplasmic machinery. For optimal staining results, the EA 50 stain should have properties that are in complete compliance with other reagents used in the Papanicolaou staining process.

Precautions

• The reagent is highly flammable.
• Keep it away from the sparks, heat, hot surfaces, open flames, and other ignition sources.
• Wear eye protection, gloves, and protective clothing.
• Causes serious eye irritation. In the case of exposure, rinse the eyes cautiously with water for several minutes.
• Causes damage to organs if swallowed.
• Store in a tightly closed container, and keep it away from bright light.

Summary

• Eosin Azure (EA) 50 is a counterstain used for polychromatic cytological staining of gynecological samples.
• Papanicolaou stain comprises of both the basic and acidic dyes. Basic components of the dye stain the acidic constituents of the cell while the acidic part stains the basic components of the cell.
• Papanicolaou cytological staining technique is a standard technique for hormone cycle and cancer diagnosis. It imparts a range of coloration to exfoliative cells of vaginal body secretions enabling the examination of the cellular constituents.

References

1. Carson, Freida L; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.) Hong Kong: American Society for Clinical Pathology Press. Pp.361-3363. ISBN 978-0-89189-581-7.
2. Murphy, M. W. (1990). Current status of urinary cytology in the evaluation of bladder neoplasms. Hum Pathol, 21(9), 886-896.
3. Papanicolaou, G.N. (1941): Some improved methods for staining vaginal smears. JLab Clin Med.