Histosonda®

INTRODUCTION

c-erbB2 is denominated as such as it corresponds to the human homologue of a viral gene that causes leukemia erythroblastosis in birds.  This gene was isolated in a neuroblastoma/glioblastoma  rat cell colony and for this reason it is also known as HER2-NEU.

This gene codes for a tyrosine kinase receptor situated in the cytoplasmic membrane for whom the ligand is still unknown.

This protein belongs to a family of receptors whose principle member is EGFr (epidermal growth factor receptor) also known as c-erbB1.  This family is made up of four members (c-erbB1, c-erbB2, c-erbB3 and c-erbB4) that share great internal homology.  Due to this homology antibodies targeted against this family of proteins can cross react with other members.

It is clinically shown that between 15 and 30% of breast cancers over-express the c-erbB2 protein.  This over-expression implies a worse prognosis for these tumors meaning that they are more aggressive and resistant to Taxol.

As there are therapeutic monoclonal antibodies against this protein is it vital to know if a concrete tumoral case over-expresses the c-erbB2 protein or not.

Until now the methods of detection have been based on immunohistochemistry using antibodies targeted against the c-erbB2 protein (which generally yield confusing results) and the determination of genetic amplification of the gene that codes for this product.

Cenbimo has designed a probe for in situ hybridization that detects the transcribed RNA of the c-erbB2 gene.

The detection of messenger RNA for this protein offers multiple advantages as it eliminates the confusing aspects of immunohistochemistry, and detects real transcription, as to see genetic amplification by FISH does not guarantee that all amplified genes are transcribing.

Histosonda c-erbB2 consists of 2 fragments of single-stranded DNA with lengths of 672 and 1143 nucleotides that are targeted against expressed sequences of this gene.

The probe incubation period is only 1 hour and as it is a chromogenic hybridization probe the results can be viewed directly by light microscope.

INTENDED USE

For use in In Vitro Diagnosis.

The Kit Histosonda c-erbB2 is useful for the detection of c-erbB2 RNA and its associated tumors.

WARNINGS AND PRECAUTIONS

The Kit Histosonda c-erbB2 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 c-erbB2; 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 c-erbB2 consists of two segments of single-stranded DNA complementary to expressed RNA with lengths of 672 and 1143 nucleotides.  The DNA of this probe has 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 c-erbB2 RNA 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 manufacturer's laboratories using 20 year old paraffin blocks with optimal results.

INTERPRETATION OF RESULTS

Samples in which c-erbB2 expression is observed will show a brownish color in the cell cytoplasm, 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 c-erbB2 has been optimized to detect c-erbB2 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 the 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 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.

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