PCR - based Clonality Analysis in Diffuse Large B-cell Lymphoma Using BIOMED-2 Primers of IgH (FR3) on Formalin-fixed Paraffin-embedded Tissue

BIOMED 2 multiplex polymerase chain reaction (PCR) protocol is a widely accepted tool for evaluation of clonality in lymphoma diagnosis. Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin,s lymphoma and displays a special challenge for PCR-based clonality analysis due to a high frequency of somatic hypermutation in rearranged immunoglobulin (Ig) domains. In this study, we evaluated detection of B- cell clonality in DLBCL by using Ig heavy chain (IgH) framework region 3 (FR3) primers in formalin- fixed paraffin- embedded (FFPE) tissue. FFPE samples from 100 cases diagnosed as DLBCL in the period of 2005 through 20011 were assessed in this study. Clonality of IgH (FR3) was evaluated by PCR amplification method that was optimized for FFPE tissue. The clonal detection rate was (62.8 %) with IgH (FR3) assay after modification, using filter for better DNA purification on negative cases. DNA quality in FFPE samples stored in recent five years were significantly better than older paraffin blocks (P < 0.001). Although a higher rate of clonality was observed in more recent group, it was not statistically significant. By using IgH (FR3) primers followed by one additional filter tube for better DNA purification, we could achieve a considerable rate of clonality with little adverse impact of DNA degradation.