Limitations of clonality analysis of B cell proliferations using CDR3 polymerase chain reaction

Background/Aims-Detection of clonal immunoglobulin heavy chain (IgH) rearra ngements by the polymerase chain reaction (PCR) is an attractive alternativ e to Southern blotting in lymphoma diagnostics. However, the advantages and limitations of PCR in clonality analysis are still not fully appreciated. In this study, clonality was analysed by means of PCR, focusing in particul ar on the sample size requirements when studying extremely small samples of polyclonal and monoclonal lesions. Materials/Methods-High resolution complementarity determining region 3 (CDR 3) PCR was used to investigate the minimum number of cells and the amount o f tissue required for the detection of a polyclonal population, both for fr esh cells and formalin fixed, paraffin wax embedded tissue. Subsequently, f rozen and paraffin wax embedded samples of 76 B cell lymphoproliferative di sorders, 43 of which were tested by means of Southern blotting, were analys ed to establish the sensitivity of this assay. These specimens included 12 chronic lymphocytic leukaemias (CLLs), nine mantle cell lymphomas (MCLs), 1 0 follicular lymphomas (FLs), and 45 mucosa associated lymphoid tissue (MAL T) lymphomas. The specificity was tested on reactive lymph nodes (n = 19), tonsils (n=4), peripheral blood lymphocyte fractions (n=4), and biopsies wi th gastritis (n = 21). Results-In reactive tissue, 20 ng of high molecular weight DNA derived from 6.5-9 x 10(3) B cells was sufficient to obtain a polyclonal PCR result. Wi th smaller amounts "pseudoclonality" could be induced. When using paraffin wax blocks, undiluted DNA isolated from tonsillar tissue of at least 1 mm(2 ) was necessary to obtain a polyclonal pattern. The sensitivity required to detect clonality in paraffin wax embedded and frozen tissue by PCR for n ( 40% and 60%, respectively) was lower than that for,1 MALT lymphomas (60% an d 86%, respectively), CLL (78% and 89%, respectively), and MCL (88% and 100 %, respectively). PCR specificity was 96% and 100% for frozen and paraffin wax embedded tissue, respectively. Conclusion-The minimum amount of template for CDR3 PCR is approximately 20 ng of high molecular weight DNA or 1 mm(3) of B cell rich paraffin wax embe dded normal tonsillar tissue, but care has to be taken to avoid pseudoclona lity when low numbers of B cells are present. Duplicate or triplicate tests should be performed to avoid misinterpretation. The specificity of the PCR assay is almost 100%, whereas sensitivity depends on a combination of fact ors, such as lymphoma type and tissue fixation. Because frozen samples yiel d better results, obtaining fresh material for the PCR assay is recommended , especially when analysing FL and MALT lymphomas.