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EA-65 Stain



‘-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 non-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



Eosin Azure 65 is a cytological counterstain used for non-gynecological cytodiagnosis in Papanicolaou staining technique. The cells to be examined in cytodiagnosis could be of animal, human or plant origin. In human cytodiagnosis, the experiment can be further categorized into gynecological and non-gynecological. Non-gynecological samples are collected from urine, sputum, bronchial secretion, or ascites that contains squamous epithelial cells or other such cells. Through cytodiagnosis, the specimen collected can be examined for the presence of premalignant and malignant cells. After this examination, the cells are classified as normal, suspected, inflammatory, uncertain or malignant cells.


Dr. GN Papanicolaou first developed the Papanicolaou stain in 1947, and since then it has been successfully used for cervical cancer diagnosis. It has also reduced the incidence of cervical cancer by 70% particularly in developed countries allowing the well-planned screening procedures. However, the stain has been modified from the conventional regressive method to progressive rapid Pap staining to reduce the duration of the procedure. Later the other modifications were Enviro- Pap stain to make the dye environment-friendly; REAP stain which was rapid and economical, and Cytocolor in which isopropyl alcohol was replaced by Propanol.

Papanicolaou defined the main objectives of staining the cytological smears as follows:

Cellular differentiation  

The differentiation of cells is important for the identification of the different cell types found in cytological smears.  

Nuclear detail definition

Since there are many nuclear abnormalities in cancerous cells, appropriate 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.


The Pap staining procedure is used to examine cells in smear preparations. The EA 65 stain is used to examine non-gynecological preparations or gynecological preparations.

Examination of cellular and humoral responses to gene therapy (Y. Dai. et al., 1995)

The EA 65 staining has also been used to examine the humoral and immune response to adenoviral vector gene therapy. In the study, recombinant adenoviruses containing the canine factor IX (FIX) cDNA were introduced in the leg muscle of the test animals. The tissue sections of the target muscles were counterstained with Eosin Azure 65 for morphological examination and expression assessment. High expression (1-5 ug/ml in plasma) of FIX protein was detected for >300 days in nude mice. Whereas, the expression of FIX protein was transient (7-10 days) in normal mice. It was also presented that the CD8+ lymphocytes could be detected within 3 days in the infected muscle tissue. It was found that the normal mice, once infected with recombinant adenovirus, could not be reinfected for at least 30 days because of the presence of neutralizing viral antibodies. With the use of immunosuppressive drugs, some normal mice can be tolerized to secrete FIX protein for >5 months. It was concluded that the adenoviral vectors have some limitations for long-term gene therapy and in the future, they could be further researched and advanced.

Cervical Acid Phosphatase-Papanicolaou (CAP-PAP) Test (Markovic., Markovic., & Belledonne., 1999)

The Papanicolaou staining technique is a gold standard method for cervical cancer diagnosis, staging, and treatment monitoring. The cervical acid phosphatase-Papanicolaou test (CAP-PAP) test is a simple, rapid, and inexpensive procedure that can alleviate the issue of false negatives. CAP speeds up the liberation of phosphate from alpha-naphthyl AS-BI phosphate. The remaining aromatic part simultaneously couples with fast garnet GBC and produces an insoluble brown-red diazonium salt on the sites of the enzyme activity. The cytoplasm is counterstained with OG and EA solutions, and the nuclei are counterstained with hematoxylin solution. The procedure allows simultaneous assessment of the CAP activity and the cellular morphology.

Strengths and Limitations

EA-65 Stain is a stable and reliable dye for the identification and examination of non-gynecological cells for neoplasia and cancer. The dye reacts exclusively with the intended cells aiding in their identification and accurate diagnosis after examining the smear preparation. The diagnostic tests are not always accurate and at times could lead to false results.


  • EA-65 Stain it is a highly flammable liquid keep it away from spark and hot surfaces.
  • May cause eye and skin irritation, seek medical treatment in case of contact.
  • Do not breathe vapors, spray or mist as it may cause target organ toxicity if inhaled.
  • Do not smoke, eat or drink when using this product.


  • Papanicolaou (Pap) staining is a cytological technique used to differentiate cells in smear preparations.
  • The technique is reliable for cell differentiation from various bodily secretions such as urine, abdominal fluid, gynecological smears, tumor samples, and seminal fluid.
  • EA-65 Stain is a significant component of the Papanicolaou stain, a procedure that is useful in cancer screening and diagnosis.


Papanicolaou, G.N. (1941): Some improved methods for staining vaginal smears. J Lab Clin Med.

Conventional Pap Smear and Liquid Based Cytology for Cervical Cancer Screening – A Comparative Study, Journal of Cytology, 24 (4): pp 167-172. Instructions OG6-OT-100 OG6-OT-110 OG

  1. Markovic., N. Markovic., & Belledonne., M. (1999). Cervical Acid Phosphatase-Papanicolaou (CAP-PAP) Test. Journal of Histotechnology, 22(1).
  2. Dai., M. E. Schwarz., D. Gu., W. Zhang., N. Sarvetnick., & Verma., M. (1995). Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci U S A, 92(5), 1401-5.


Additional information


1 Gallon, 16 oz set of 4

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