CENBIMO S.L

INTRODUCTION

The Epstein-Barr virus is a member of the gamma-herpes viruses (HHV-4). It is a linear 184,000 base pair double stranded DNA virus and was the first virus to be discovered as oncogenic (1).

Primoinfection by this virus can show signs of a slight viral infection or it can present as Infectious Mononucleosis. The most common target cells for the Epstein-Barr virus are the B lymphocyte and nasopharyngeal epithelial cell.

The Epstein-Barr virus massively infects the human population and sero-epidemiological studies show that 90% of adults have been infected by this virus (2).

Latently infected B lymphocytes express abundantly (104 -105 copies) among other genes, a short nonpolyadenylated chain of RNA that does not transduce to a protein, consisting of two fragments know as EBER 1 and EBER 2. The expression of EBER (Epstein-Barr virus encoded RNAs) is nuclear. Although the function of EBER is unknown, it is believed that it may play a role in virus-produced oncogenesis (3).

There are numerous human tumors associated with EBV, ranging from non-differentiated nasopharyngeal carcinoma to African Burkitt's lymphoma, Hodgkin's disease Mixed cellularity to some B, T and NK lymphomas, as well as lymphoproliferative processes associated with immunodeficiency(4).

INTENDED USE

For use in In Vitro Diagnosis

The Kit Histosonda EBER is intended for the detection of EBER 1+2 RNA in cells infected by the Epstein-Barr virus, in both reactive and tumoral cells. Its use is intended for the aforementioned tumors, and also in lymph nodes of patients with atypical reactive pathology as produced by Infectious Mononucleosis. This detection is carried out by in situ hybridization in paraffin-embedded, formalin-fixed histological sections.

WARNINGS AND PRECAUTIONS

The Kit Histosonda EBER has been designed for professional use in In Vitro Diagnosis and must be manipulated by qualified and accordingly trained personnel.

In order to obtain the best results, the instructions contained in the manual must be followed. Any change to the indicated temperatures, times or any other step of the process can lead to poor results.

KIT COMPONENTS

The Kit includes 20 single test tubes of Histosonda EBER; 20 single test tubes of Proteinase K and 2 tubes of Anti-Digoxin antibody sufficient for 20 tests in total.  All products are lyophilized.

Histosonda EBER consists of a fragment of single stranded DNA of 526 nucleotides, complementary to expressed EBER. This probe detects the EBER gene in full, including EBER 1 and EBER 2. The DNA of the probe contains nucleotides that have been labeled with Digoxigenin.

STORAGE CONDITIONS

Supplied reagents are stored at room temperature until their expiration date. After being reconstituted the probes will remain stable for two weeks at 4ºC in a DNAase-free environment.  The Anti-Digoxin can be stored at 4ºC for 1 month and the Proteinase K must be used immediately and cannot be stored.  Do not use these products after their expiration dates.

SAMPLES

Any paraffin block section in which EBER presence is to be studied. Sections of 4-6 micrometers in width are sufficient to conduct the study. Preferably, the cut should be recent (no more than thirty days old). Assay results are not affected by block age. Studies have been carried out in the manufacturers’ laboratories using 20 year old paraffin blocks with optimal results.

INTERPRETATION OF RESULTS

Samples in which EBER expression is observed will show a brownish color in cell nucleus, which will contrast over the blue-violet background given by hematoxylin staining. The pathologist will evaluate the results according to their experience, drawing conclusions from the staining of the sample in parallel with the staining observed in positive and negative controls.

ASSAY LIMITATIONS

The Kit Histosonda EBER has been optimized to detect RNA expression in formalin-fixed, paraffin-embedded tissues. Its use is not recommended for other types of samples or preparation techniques.

The correct operation of this kit has been validated using the protocols indicated in the instructions manual. The use of other procedures or modification of the recommended protocols may lead to erroneous results. The results from this assay must be evaluated by the pathologist in combination with the rest of the available patient clinical data.

In order to obtain optimal and reproducible results it is important to rigorously maintain the time and temperature conditions indicated in the procedure.

BIBLIOGRAPHY

1) Epstein M, Achong B, Barr Y. Morphological and biological studies on a virus in cultured lymphoblast from Burkitt's lymphoma. J.Exp.Med. 1965; 121:761-770.

2) Henle G, Henle W, Clifford P, et al. Antibodies to EB virus in Burkitt's lymphoma and control groups. J. Nat. Cancer Inst. 1969; 43: 1147-1157.

3) Komano,J,S. Maruo,K. Kurozumi,t. Oda,K. Takada. 1999. Oncogenic role of Epstein-Barr virus encoded RNAs in Burkitt's lymphoma cell line Akata. J. Virol 73: 9827-31

4) Elaine S. Jaffe, Nancy Lee Harris, Harald Stein, James W. Vardiman. 2001. Tumors of Haematopoietic and Lymphoid Tissues (World Health Organization Classification of Tumours). IARC Press. Chapters 6,7,8.