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

Abstract

Background/aims: Detection of clonal immunoglobulin heavy chain (IgH) rearrangements by the polymerase chain reaction (PCR) is an attractive alternative 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 particular on the sample size requirements when studying extremely small samples of polyclonal and monoclonal lesions.

Materials/methods: High resolution complementarity determining region 3 (CDR3) PCR was used to investigate the minimum number of cells and the amount of tissue required for the detection of a polyclonal population, both for fresh cells and formalin fixed, paraffin wax embedded tissue. Subsequently, frozen and paraffin wax embedded samples of 76 B cell lymphoproliferative disorders, 43 of which were tested by means of Southern blotting, were analysed to establish the sensitivity of this assay. These specimens included 12 chronic lymphocytic leukaemias (CLLs), nine mantle cell lymphomas (MCLs), 10 follicular lymphomas (FLs), and 45 mucosa associated lymphoid tissue (MALT) lymphomas. The specificity was tested on reactive lymph nodes (n = 19), tonsils (n = 4), peripheral blood lymphocyte fractions (n = 4), and biopsies with 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. With smaller amounts "pseudoclonality" could be induced. When using paraffin wax blocks, undiluted DNA isolated from tonsillar tissue of at least 1 mm2 was necessary to obtain a polyclonal pattern. The sensitivity required to detect clonality in paraffin wax embedded and frozen tissue by PCR for FL (40% and 60%, respectively) was lower than that for MALT lymphomas (60% and 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 mm3 of B cell rich paraffin wax embedded normal tonsillar tissue, but care has to be taken to avoid pseudoclonality 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 factors, such as lymphoma type and tissue fixation. Because frozen samples yield better results, obtaining fresh material for the PCR assay is recommended, especially when analysing FL and MALT lymphomas.

Figure 1

Results of the complementarity determining region 3 (CDR3) polymerase chain reaction. Lanes 1–6, lymphoma case (lanes 1 and 2, 200 ng DNA from frozen tissue, showing a clonal band in a polyclonal background; lanes 3–6, DNA from paraffin wax embedded tissue, undiluted and 1/10, 1/100 and 1/1000 dilutions, respectively, giving the same clonal band in the three diluted samples); lane 7, water; lane 8, Jurkatt T cell line; lane 9, peripheral blood lymphocyte fraction, showing a polyclonal ladder of the expected size range; lane 10, positive control (chronic lymphocytic leukaemia (CLL) DNA); lane 11, positive control (CLL DNA mixed with DNA from an Epstein-Barr virus infected cell line).

Figure 2

Complementarity determining region 3 (CDR3) polymerase chain reaction on a dilution series of DNA isolated from a frozen sample of a reactive tonsil. Lane 1, 200 ng DNA, showing a normally distributed polyclonal ladder; lanes 2–11, 10 fold dilution steps producing oligoclonal patterns with randomly (dis)appearing bands.