| Abstrakt: |
PCR protocols for immunoglobulin heavy chain (IgH) gene rearrangements amplification make easy the NHL-B identification. In this study we analyzed PCR products by Capillary Electrophoresis (CE) and GeneScan (GS) software, wich offers clear advantages over the conventional methods such as agarose gels (AGGE), characterized by hight rate of false negative and false positive results. We suggested some criteria--not included in previous NHL-B issues--useful to a correct analysis of results in GS. Since 2003, we collected 2,977 samples (2,770 peripheral blood and bone marrow, and 207 tissues) for GS analysis from NHL-B patients. At beginning PCR products were detected by both AGGE and CE. FR2 and FR3 VH regions were amplified by PCR seminested; together with Bcl-6 "housekeeping" gene from the same sample, as marker of DNA quality and PCR efficiency. Bcl-2/IgH and Bcl1/IgH traslocations were also analyzed for follicular and mantle cells lymphomas respectively. Resolution and sensitivity tests, developed with serial diluitions of clonal products in water and in DNA from healthy individuals, showed for GS 1% of resolution limit (3% AGGE) and 0.5% of sensitivity (5% AGGE). Our criteria for correct interpretations of results are: a) use of "house-keeping" gene Bcl-6; b) costant reference scales for hight and molecular weight; c) clonal peak at least twice higher than adiacent peaks; d) position of clonal peak (central or eccentric) as regards to policlonal peaks distributions. e) peaks features for oligoclonal or biallelic rearrangements evaluation. GS is an ideal method for detecting IgH rearrangements and some characteristic traslocations. The precise determination of the size of the PCR product can be used for the minimal residual disease evaluation. Moreover, it allows semi-quantitative resolution of fragments only one base different in size and may be more objective than gel-based methods. |
