EA-36 Stain – Giemsa Stain

$99.90$269.90

-Provides excellent cytoplasmic staining of gynecological and non-gynecological samples

-Used in conjunction with Hematoxylin nuclear stains in the diagnosis of malignant cytological diseases

-Used in conjunction with OG-6 for gynecological staining

-Our papanicolaou cytology stains are available in a wide range of formulations to allow the end user to select from various colors intensities and hues

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Introduction

Eosin Azure (EA 36), also known as Papanicolaou stain, is a counterstain used to differentiate cells in various bodily secretions. The specimens could be gynecological smears, urine, sputum, cerebrospinal fluid, pleural fluid, synovial fluid, abdominal fluid, seminal fluid, fine needle aspiration, tumor samples, or other samples containing cells. The biological and physiological behavior of cells can be interpreted qualitatively and quantitatively in terms of growth activity and functional differentiation using the EA 36 stain. Unstained cells are practically invisible under a microscope; therefore, the cells are stained for easier interpretation of cytomorphological patterns. The nucleus of the cell indicates normal or abnormal growth activity while the nucleolus indicates the functional differentiation of a cell. Differential counterstaining is the prime objective of the Papanicolaou staining which is achieved by the application of OG-6 and EA stains.

Papanicolaou EA-36 comprises of 3 dyes: EA -36, EA -50 and EA -65 with the number denoting the proportion of the dye in the mixture. EA -36 and EA -50 are used in gynecological staining while EA -65 is specific for non-gynecological staining. Papanicolaou EA 36 stain is used in conjunction with hematoxylin nuclear stains for vaginal smears to diagnose any malignant cytological condition (vaginal, cervical and uterine cancer). It is used during the gynecological examination to stain and examine the cellular morphology especially for cervical cancer detection and other pathological changes before the disease development. With this staining procedure, the hormonal levels could also be analyzed, and any pathological organism such as trichomonas and candidiasis in genital meatus can be also be detected.

Composition

  1. Light green; 45.0 gm
  2. Bismark brown; 10.0 gm
  3. Eosin Y; 45.0 gm
  4. Phosphotungstic acid; 0.20gm
  5. Lithium carbonate, saturated aqueous solution; 1drop.

The formula of the stain can be adjusted and standardized for the specific performance parameters.

History

The Papanikolaou stain was discovered by a Greek scientist named as Georgios Papanikolaou in 1913. He is considered as a pioneer in the field of cytopathology and has made enormous contributions to early cancer detection particularly cervical cancer.  In 1928, he was the first to suggest that a noninvasive technique can be used to gather cellular debris from the lining of the vaginal tract for microscopic examination. The importance of his research was recognized in 1943 when he along with Herbert F. Traut, an American gynecologist, and obstetrician, published their book: Diagnosis of Uterine Cancer by the Vaginal Smear. They covered the topics including the preparation of cervical smears, the cytological and physiological changes during the menstrual cycle, the changes detected in the presence of cervical cancer and the effects of related pathological conditions.

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Principle

Eosin Azure (EA 36) is an alcoholic solution of glacial acetic acid, eosin Y, phosphotungstic acid and Fast Green (FCF). Phosphotungstic acid acts by selectively excluding the eosin from the cytoplasm of specific cells facilitating the staining by Fast Green FCF. Sites that remain unstained by Fast Green FCF, orange G, and hematoxylin are then stained by eosin. Acetic acid works by further acidification and facilitates the Fast Green FCF and eosin staining.

Procedure

Specimen Preparation

Flattened cells are preferred for optimal display and staining. Unfixed fresh cells are normally collected on a membrane filter, spread ideally as a monolayer on a slide and immediately wet-fixed in 95% ethyl alcohol. Non-flattened cells normally use more stain and limit microscopic examination. Non-flattening in cells can be attributed to:

  • Thick spreads.
  • Spreading on albumenized slides
  • Collection in preservative
  • Spray fixation
  • Suspension in a mucus stream
  • Part of a tissue fragment.

To correct this one of the following alternatives can be tried:

  • Ethyl alcohol
  • 90% acetone
  • Absolute methyl alcohol
  • 80% isopropyl alcohol
  • 95% reagent grade alcohol

Fixation

Fix the smears in 95% alcohol for 5-15 minutes. Avoid prolonged fixation as it may have negative effects on the staining reaction.

Staining

  1. Rinse the smear with 70% alcohol, 50% alcohol and then finally with the distilled water.
  2. Stain the smear in Harris hematoxylin for 5-10 minutes.

Note: This should be done in the absence of acetic acid.

  1. Rinse the smear with distilled water.
  2. Wash the smear 3 or 4 times with 0.5% aqueous solution of HCL.
  3. Soak it in lithium carbonate solution for 60 seconds. The solution concentration should be low. Then rinse it with water thoroughly.
  4. Again rinse it in distilled water and increasing alcohol concentrations of 50%, 70%, 80%, and 95%.
  5. Stain the smear in Papanicolau OG 6 stain for 60 seconds.
  6. Rinse 5-10 times in 95% alcohol.
  7. Stain in Eosin Azure 36 stain for 2 minutes.
  8. Rinse 5-10 times again in 95% alcohol.
  9. Rinse in absolute alcohol.
  10. Rinse in equal parts of absolute alcohol and then in xylene.
  11. Mount the smear in a satisfactory medium and observe it under a microscope.

Expected Results

If the smear is stained correctly, it should exhibit hues of the entire spectrum (red to violet). Following results are presented after the EA 36 staining;  

  • Erythrocyte: Salmon pink or vermeil.
  • Epithelia: nucleolus-red, the nucleus-indigo.
  • Keratinized cells appear pink.
  • Cells before keratinization are sky-blue or light green while the full-keratinized cells appear orange.
  • Mucus: sky-blue or pink.
  • Leukocyte:  nucleolus-indigo, cytoplasm-sky-blue

A well-prepared smear will display a crisp blue with black cell nuclei. The cytoplasm displays a pink to pale pink color. Superficial cells are orange to pink while glycogen and cells with high keratin content are yellow. Metaplastic cells often stain both green and pink at once. Intermediate and parabasal cells are turquoise green to blue.

Applications

Histological examination of the placenta (Difruscio. & Desa., 1977)

Since its introduction, the Papanicolaou staining technique has gained universal acceptance in the field of cytology and histology. In routine vaginal cytological preparations, the Papanicolaou stain has enabled the detection of monilia species and a number of different organisms. It provides excellent differential staining of both the nucleus and the cytoplasm. Chorioamnionitis is one of the abnormalities found in placental specimens. The organisms responsible for chorioamnionitis are usually present in the vagina. Therefore, EA 36 staining is a suitable technique for placental histology.

Cervical cancer diagnosis (Mishra., Pimple., & Shastri, 2011)

The Papanicolaou stain has a wide array of applications in cervical cancer diagnosis. The method has been modified to improve the specificity, sensitivity, and reproducibility. Cervical cancer can be prevented through identifying and treating the precancerous lesions; therefore, preventing the disease progression to cervical carcinoma. A variety of visual characteristics, such as morphology, shape, and the optical density of the cells, are examined for the presence of malignancy. The Papanicolaou stain has enabled the early detection and prevention of cervical cancer.

Cytology of various organs (Shinde. & Pandit., 2006)

The Papanicolaou stain has been modified to minimize the staining time. Of these modifications, ultrafast Papanicolaou stain technique has been employed on fine-needle aspiration cytology (FNAC) smears of the breast, ovary, thyroid, bone, lymph node, central nervous system, and lungs. The smears were evaluated for the smear background, staining pattern, cell morphology, and nuclear characteristics. With manifold advantages of the Papanicolaou stain, it could be considered as a reliable and useful staining option for rapid diagnosis of carcinomas as well as for intraoperative cytology.

Strengths and Limitations

Merits of the EA stain

The Eosin Azure 36 stain is stable in solution with predictable, high-quality staining results. It is a gold standard technique for gynecological studies to detect abnormal cells and any malignancy.

 

Limitations of the EA 36 dye

The EA 36 stain is a bit more complex with a complicated history as compared to the OG-6 stain. The EA formula is obscure with many variations at times resulting in unsatisfactory staining.  

Precautions

  • The stain should be stored in a tightly closed container below 30°C.
  • Do not smoke, eat or drink when handling the staining solution.
  • Extremely flammable! Keep the stain away from heat, hot surfaces, sparks or open flames.
  • Use it only in outdoors or in a well-ventilated environment.
  • Wear protective clothing- gloves and eye protection when using this product.

Summary

  • EA 36 is mainly intended for the gynecological specimen as an essential ingredient in the Papanicolaou stain.
  • The Papanicolaou staining method is a useful method for early cancer detection and prevention.
  • EA 35 is a common ingredient of the Papanicolaou method used for the staining of exfoliated cytological specimens.

References

B. P. Shinde., & Pandit., A. A. (2006). Application of modified ultrafast Papanicolaou stain in cytology of various organs. Diagn Cytopathol, 34(2), 135-9.

Carson, Freida L; Hladdik, Christa (2009). Histotechnology: A Self-Instructional Texx (3 ed.). Hong Kong: American Society for Clinical Pathology Press. Pp. 361-3363. ISBN 978-0-89189-581-7.

D. Difruscio., & Desa., D. J. (1977). Application of the Papanicolaou stain to routine histological examination of the placenta. J Clin Pathol, 30(7), 682-683.

Frost, J.K., The cell in Health and Disease, Williams and Wilkins, Baltimore, 1969.

G. A. Mishra., S. A. Pimple., & Shastri, S. S. (2011). An overview of prevention and early detection of cervical cancers. Indian J Med Paediatr Oncol, 125-132.

Papanicolau, G.N., 95, 438-439 (1942).

Papanicolaou, G.N., Atlas of Exfoliative Cytology, published for the commonwealth Fund by Harvard University Press, Cambridge, 1954, p. 6.3.

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    Size

    16 oz set of 4, 4 Gallons