PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number

Abstract

Polymerase chain reaction (PCR)-based analysis for detecting immunoglobulin heavy chain gene (IgH)rearrangements in lymphoproliferative disorders is well established. The presence of one or two discrete bands is interpreted as a monoclonal proliferation, whereas a smear pattern represents a polyclonal population. Prompted by our observation of discrete bands in histologically reactive processes with a relative paucity of B cells, we sought to determine whether low numbers of B cells in biopsy specimens could artifactually produce pseudomonoclonal bands. We performed IgH PCR analysis on serially diluted DNA samples from 5 B cell non-Hodgkin's lymphomas (B-NHLs), 5 reactive lymph nodes, 5 reactive tonsils and 10 microdissected germinal centers from a lymph node with follicular hyperplasia. We also assessed multiple aliquots of DNA samples from small biopsy specimens of reactive lymphocytic processes from the stomach (5 cases). PCR products were evaluated using high resolution agarose or polyacrylamide gels, and DNA sequencing was performed on IgH PCR products from two reactive germinal centers, which yielded monoclonal bands of identical size. The utility of molecular techniques such as Southern blot hybridization (SBH) analysis and polymerase chain reaction (PCR) in the assessment of clonality in lymphoproliferative disorders is well established. Although SBH is a fairly sensitive and specific method, its utility as a routine diagnostic tool is hampered by its requirement for high quality DNA, its labor-intensive nature, and its consequent long turnaround time. On the other hand, the rapid and less labor-intensive PCR continues to gain popularity as a technique for the assessment of clonality in lymphoproliferative disorders. 1,2 The results obtained with antigen receptor gene PCR correlate well with the results of SBH. [3][4][5] PCR-based assays have additional advantages over SBH analysis. PCR requires smaller quantities of DNA, and high molecular weight DNA is not necessary. Therefore, PCR has been applied to small biopsies and fixed paraffin-embedded tissues, which are generally unsuitable for SBH analysis. 6 PCR is extremely sensitive and can detect 1 positive cell in a background of 10 5 negative cells. Immunoglobulin heavy chain (IgH) PCR is capable of detecting 1 monoclonal B cell in a background of 10 2 to 10 3 polyclonal B cells. 7 The extreme sensitivity of PCR also constitutes a potential source for pitfalls in the interpretation of PCR-based antigen receptor gene rearrangement studies. Clearly, contamination is a frequent concern. Additionally, we have observed discrete bands in samples obtained from small biopsy specimens in which histological and immunophenotypic evaluation revealed a reactive process. We believe that the discrete bands generated in this situation are related to the paucity of B

Nasal-type extranodal natural killer/T-cell lymphomas: a clinicopathologic and genotypic study of 42 cases in Singapore

“…The exact clinical significance of these 'pseudospikes' or 'oligoclonal peaks' in NK/TCL remains unascertained and it is unclear whether they are a result of tumor heterogeneity, random amplification of a low number of T cells in the samples, or related to the frequently abundant background polyclonal T-cell targets present in NK/TCL, which effectively compete with the monoclonal population during amplification. 40 Mutation of the p53 gene is frequently implicated in the pathogenesis of many human cancers. The wild-type protein is normally present in cells at undetectable levels because of its very short halflife.…”

PCR Methods for Determining B Cell Clonality

“…Sabath, Wood, and Kussick in our recent article. 1 Our study highlighted the potential for incorrectly assigning monoclonal status to samples containing polyclonal or oligoclonal B cell populations. In our study, we compared hemi-nested and non-nested single-step PCR approaches followed by polyacrylamide gel electrophoresis.…”

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We read with interest the article by Elenitoba-Johnson et al, 1 in which the authors demonstrated potential false positive results obtained using a polymerase chain reaction (PCR)-based method to determine B cell clonality. This is an important observation, and one must be cognizant of the potential for detecting clones of uncertain clinical significance in specimens with very few B cells.

However, we would like to make the observation that the source of this problem is the hemi-nested PCR method used by the authors, which is compounded when only small amounts of DNA are used in the assay.

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“…We read with interest the article by Elenitoba-Johnson et al, 1 in which the authors demonstrated potential false positive results obtained using a polymerase chain reaction (PCR)-based method to determine B cell clonality. This is an important observation, and one must be cognizant of the potential for detecting clones of uncertain clinical significance in specimens with very few B cells.…”

“…While we state that "the hemi-nested assay is more prone to the generation of pseudo-monoclonal bands" in agreement with their experience, we also demonstrate that this phenomenon may occur, albeit less frequently, in non-nested assays. 1 Recognition of this phenomenon is particularly important when the DNA source is fixed paraffin-embedded tissue, where the DNA quality may be suboptimal. This technically diminishes the DNA target that is available for amplification.…”