A New Non-Radioactive Method For Detection of Monoclonal Cell Populations in Patients With Burkitt’s Lymphoma
We developed a new non-radioactive method to investigate the clonality of biopsy material, bone marrow samples or peripheral blood mononuclear cells from children with Burkitt’s lymphoma, using the polymerase chain reaction (PCR).
The variable region (VH) of the immunoglobulin heavy chain (IgH) was used as the clonal marker in B-lymphoproliferative disease.1 For amplification of the IgH variable region, we used 7 VH region family specific primers and one joining region (JH) consensus primer in one PCR reaction.2,3,4 The JH primer was labeled at the 5’ end with digoxigenin. After PCR, the products were separated under denaturing conditions in a 4% polyacrylamide gel on a direct blotting apparatus (GATC, MWG Biotech, Ebersberg, Germany). During electrophoresis, the blotting membrane under the gel was not transported until the dye markers indicated a product length of approximately 300 bp. The membrane was then transported for 120 min. These conditions allow for wide separation of the expected products of 300–400 bp length. After blotting, the membrane was crosslinked by UV-light, blocked and incubated with an alkaline phosphatase conjugated anti-digoxigenin antibody. After washing, the specific PCR products were detected by the chemiluminescent substrate CSPD and the membrane was exposed to x-ray film. With this method we were able to differentiate between monoclonal and polyclonal material at a sensitivity of 2% clonal cells mixed into peripheral blood mononuclear cells. The specific advantage of this method is that the same membrane can be used for hybridization with a clone-specific oligonucleotide probe. This allows characterization of the specific PCR-product by hybridization and by size fractionation at the same time, and it increases specificity and sensitivity. The method could be useful to detect malignant cells in the peripheral blood of Burkitt’s lymphoma patients during theraphy.
KeywordsLymphoma EDTA DMSO Leukemia Recombination
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