Histosonda®

INTRODUCTION

Immunologically competent B lymphocytes develop from a lymphoid stem cell until acquiring a receptor consisting of a single immunoglobulin molecule specific for a determined antigen. These immunoglobulin molecules are formed by two identical heavy chains and by two identical light chains (kappa or lambda).

In humans, in normal or reactive lymphoid tissues, approximately 60% of the B lymphocytes bear kappa light chains and the remaining 40%, lambda light chains. In B lymphocyte tumors, there is generally only one clone that proliferates, be it kappa or lambda, in a monoclonal way.

In humans the gene that codes for kappa light chains is located on chromosome 2, and the gene that codes for lambda light chains is located on chromosome 22. The variable regions for genetic recombination are located in the first 5' half of these genes, while the constant regions are located in the 3' half. There is only one functional gene for the constant region of k light region, and four functional genes for the constant region of λ light chains (Cλ1, Cλ2, Cλ3, and Cλ7).

Detection of monoclonality is one of the most important tools for differentiating B lymphoid tumors from reactive processes.

In situ hybridization technique offers an important advantage over immunohistochemistry, as it virtually lacks background, and allows a clean and sharp viewing of the histological preparation. It is also useful to differentiate cells that have absorbed immunoglobulins, and are therefore detectable by immunohistochemistry, but in fact do not produce immunoglobulin, as occurs with the Reed-Sternberg cells of Hodgkin's disease.

INTENDED USE

For use in In Vitro Diagnosis.

Histosonda Dual Kappa-Lambda is useful for the study of monoclonality in lymphoid tumors, lymphoproliferative syndromes, myelomas and for the study of immunodeficiency associated lymphoproliferative syndromes.

WARNINGS AND PRECAUTIONS

Histosonda Dual Kappa-Lambda 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.

COMPONENTS

20 single test tube of Histosonda Dual Kappa-Lambda and 20 single test tubes of Proteinase K.  All products are lyophilized.

Histosonda Dual Kappa-Lambda consists of two fragments of single-stranded DNA with lengths of 153 and 182 nucleotides that are complementary to expressed RNA. In this probe the Lambda fragment has been labeled with Digoxigenin and the Kappa fragment has been labeled with Biotin.

STORAGE CONDITIONS

Supplied reagents are stored at room temperature until expiration date. After being reconstituted the probe will remain stable at 4ºC for two weeks in a DNAase-free environment.

Do not use after expiration date.

SAMPLES

Any paraffin block section in which immunoglobulin light chains constant region 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 immunoglobulin light chains expression is observed will show color in the cell cytoplasm, which will contrast over the blue-violet background given by hematoxylin staining.  The color will be brown in the case of Lambda light chain, the color for Kappa light chains depends on the choice of chromogenic reagent used (generally red or blue). 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 the positive and negative controls for expression of this gene.

ASSAY LIMITATIONS

Histosonda Dual Kappa-Lambda 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 product 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.