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. 2002 Apr;55(2):98-101.
doi: 10.1136/mp.55.2.98.

Improvements to B cell clonality analysis using PCR amplification of immunoglobulin light chain genes

T C Diss  1 , H X Liu, M Q Du, P G Isaacson
Affiliations

Improvements to B cell clonality analysis using PCR amplification of immunoglobulin light chain genes

T C Diss et al. Mol Pathol. 2002 Apr.

Abstract

Aims: Clonality analysis using polymerase chain reaction (PCR) amplification of the immunoglobulin heavy chain (IgH) gene is an important aid to the diagnosis of B cell lymphoproliferative diseases. However, the method has a relatively high false negative rate. In an attempt to improve detection rates simple PCR strategies for clonality analysis of B cell populations using amplification of Ig light chain genes have been developed.

Methods: Novel PCR protocols, designed to amplify Ig kappa and Ig lambda light chain genes, were evaluated using high molecular weight DNA samples from 28 selected cases of B cell lymphoma with known light chain expression and 12 reactive lymphoid specimens. Products were run on 10% polyacrylamide minigels using heteroduplex analysis. Conventional IgH PCR analysis was also performed. Twelve randomly selected formalin fixed, paraffin wax processed samples from cases submitted for molecular genetic analysis were also studied.

Results: Polyclonal products were seen in all reactive lymphoid samples. Using Ig kappa PCR, 24 of 28 lymphomas, including four of five IgH negative cases, displayed monoclonal patterns. Using Ig lambda PCR, eight of 12 Ig lambda expressing tumours, including two of five IgH negative cases, showed monoclonal patterns. Standard IgH PCR demonstrated monoclonality in 23 of 28 B cell lymphomas. The detection rate was improved to 27 of 28 lymphomas using heavy and light chain PCR. Efficient amplification was achieved using paraffin wax processed samples, seven of which showed monoclonality compared with eight using IgH PCR.

Conclusions: Ig light chain PCR, used in conjunction with heavy chain analysis, enables improved detection of B cell monoclonality using routine histological specimens and can provide additional clone specific markers for the study of the biology of B cell tumours.

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Figures

Figure 1
Figure 1
Polyacrylamide gel (10%) of Igκ PCR products. Lane M, PhiXHinfI molecular weight markers; lane 1, positive monoclonal control marginal zone lymphoma (fresh tissue; FT); lane 2, follicular lymphoma (paraffin wax processed tissue; PT); lane 3, lymphoplasmacytic lymphoma (FT); lane 4, marginal zone lymphoma (FT); lane 5, follicular lymphoma (PT); lane 6, follicular lymphoma (FT); lane 7, follicular lymphoma (FT); lane 8, reactive lymph node (FT).
Figure 2
Figure 2
Polyacrylamide gel (10%) of Igλ PCR products. Lane M, PhiXHinfI molecular weight markers; lane 1, positive control lymphoplasmacytic lymphoma (fresh tissue; FT); lane 2, marginal zone lymphoma (paraffin wax processed tissue; PT); lane 3, follicular lymphoma (PT); lane 4, marginal zone lymphoma (FT); lane 5, follicular lymphoma (FT); lane 6, lymphoplasmacytic lymphoma (PT; same case as lane 1); lane 7, reactive lymph node (PT); lane 8, negative control (no DNA).
Figure 3
Figure 3
Light chain sequences derived from PCR products (primer sequences are not included). The asterisk denotes the number of identical clones with sequence shown. The nucleotide shown in lower case is the only putative somatic mutation.
See this image and copyright information in PMC

References

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    1. Diss TC, Pan L, Peng H, et al. Sources of DNA for detecting B cell monoclonality using PCR. J Clin Pathol 1994;47:493–6. - PMC - PubMed
    1. Wan JH, Trainor KJ, Brisco MJ, et al. Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction. J Clin Pathol 1990;43:888–90. - PMC - PubMed
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