Pseudo-Spikes Are Common in Histologically Benign Lymphoid Tissues

Abstract

T cell receptor gene rearrangement is a classic marker of T cell clonality and is a useful adjunct in the diagnosis of T cell lymphomas and leukemias. Rearranged V-J gene segments amplified by polymerase chain reaction (PCR) are traditionally analyzed by polyacrylamide gel electrophoresis. We and others have analyzed TCR-␥ PCR products using capillary gel electrophoresis, which produces single nucleotide resolution and provides improved diagnostic sensitivity over conventional methods. However, with this marked increase in resolution and sensitivity, it is necessary to re-define normal variation of TCR-␥ gene rearrangement in control tissues to allow appropriate interpretation of monoclonality if present. Using DNA capillary gel electrophoresis, we examined the spectrum of normal patterns for TCR-␥ in a variety of T-cell-rich, histologically benign tissue types, including spleen, lymph node, tonsil, and blood, and compared this with the patterns in T cell lymphoma samples. We defined relative peak heights as h 1 /h 2 , where h 1 represents the peak height of the largest peak above the normally distributed population, and h 2 represents the peak height of the normally distributed curve. We found spikes in almost 20% of histologically benign samples with relative peak heights that were more than 0.5 and up to 1.5. We designated these as pseudo-spikes, because they may be mistaken for monoclonal spikes. In contrast, the relative peak height of the T cell lymphoma samples that showed clonal rearrangement was much higher than that of the pseudo-spikes, being at least 2 in 11/11 and at least 3 in 10/11 cases. Our data suggest that peaks with relative height of at least 3 represent a true clonal population in diagnostic samples. Peaks with relative heights of less than 1.5 may be insignificant, while peaks with relative heights between 1.5 to 3 may warrant further evaluation. Although capillary gel electrophoresis is superior in assessing T cell clonality, caution must be exercised when interpreting results, because pseudo-spikes appear to be common in benign tissues with lymphoid populations T cells, the mediators of cellular immunity, bear T cell receptors (TCR) on their surfaces that recognize and bind to specific antigens. These receptors are unique to each T cell clone, and demonstrate great diversity through rearrangement of germline gene segments during maturation.

Cytologic evaluation of lymphadenopathy associated with mycosis fungoides and Sezary syndrome

“…32 In a study by Lee et al, up to 20% of histologically benign lymph nodes, tonsils, and spleens analyzed with TCR-c PCR showed peaks on capillary gel electrophoresis analysis that were higher than could be attributable to noise. 16 The potential for interpretative error in TCR-c PCR is only amplified in FNAB specimens, given the limited material available for molecular analysis. In all positive cases in this study, the relative peak height of the monoclonal PCR amplification product was greater than 3 times the height of the polyclonal background peaks.…”

“…Most clonal T-cell populations yield chromatographic peak heights that are [2 times that of background peak distribution. 16,33 However, given the small sample size of FNAB and the increasing reliance of cytopathologists on TCR-c PCR to establish the diagnosis of T-cell lymphoma, we believe that caution should be used when interpreting the capillary gel electrophoretic chromatographs of TCR-c PCR. For those cases in which the peak heights are 2 to 3 times that of the background peak distribution, we suggest repeating the TCR-c PCR, and if the peak is reproducible, the result can be interpreted as positive.…”

“…PCR products were denatured at 958C for 5 minutes followed by rapid cooling on ice for 5 minutes and were analyzed by capillary gel electrophoresis on an ABI 3100 instrument (Perkin-Elmer). A positive result was required to yield 1 or 2 distinct peaks within the expected size range for a given primer set with a height exceeding that of the polyclonal background by at least 2-fold to 3-fold, as described by Lee et al 16 …”

PCR Methods for Determining B Cell Clonality

“…The key recommendation emanating from our study is that diagnostic accuracy in clonality assessment may be improved by evaluating duplicate or triplicate aliquots of a sample in parallel, as has been documented by other investigators. 2 Our conclusions have broader implications for the reporting of the results of PCR-based clonality studies of the IgH 2-4 and T cell receptor genes 5,6 involving a variety of amplification and gel separation methods, including capillary electrophoresis. …”

Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations

“…Each DNA sample extracted from paraffin was tested for quality using PCR primers designed for human ␤-globin (amplicon of 264 bases determined by capillary gel electrophoresis) as described previously. 22 …”