A novel method for interpretation of T-cell receptor gamma gene rearrangement assay by capillary gel electrophoresis based on normal distribution

Abstract

T-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jgamma and four sets of Vgamma primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the chi(2) error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vgamma(1-8) is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated.

Figure 1

Common peak distributions seen in capillary gel electrophoresis of TRG clonality assays. A: Reactive, polyclonal pattern; (B) clonal peak in a polyclonal background. C: Scattered peaks without a definitive polyclonal background.

Figure 2

Representative best-fit curves (open square, idealized normal distribution; filled triangle, original data). A: Data from reactive specimens can be fitted into an idealized normal distribution curve. B: Clonal specimens have one or more peaks that do not belong to a normal distribution.

Figure 3

Data flowchart in normal distribution analysis.

Figure 4

Serial twofold dilutions of cell line DNA containing a Vγ₁ (A) and Vγ₉ (B) rearrangement (PEER) into polyclonal human genomic DNA (tonsil) were made from 1:1 to 1:255 (equivalent to 50% tumor down to less than 0.5% tumor). The DNA was amplified with the InvivoScribe BIOMED-2 T-cell clonality assay kit, separated by capillary gel electrophoresis, and analyzed with the GeneMapper software v3.7. The χ² errors (ND) are calculated by the normal distribution program described in this article. RPR and RPH are determined by the methods as described by Greiner et al and Lee et al.