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. 2007 Feb;9(1):12-9.
doi: 10.2353/jmoldx.2007.060032.

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

Frank C Kuo  1 , Dimity Hall, Janina A Longtine
Affiliations

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

Frank C Kuo et al. J Mol Diagn. 2007 Feb.

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.

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Figures

Figure 1
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
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
Figure 3
Data flowchart in normal distribution analysis.
Figure 4
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.
See this image and copyright information in PMC

References

    1. Miller JE, Wilson SS, Jaye DL, Kronenberg M. An automated semiquantitative B and T cell clonality assay. Mol Diagn. 1999;4:101–117. - PubMed
    1. Simon M, Kind P, Kaudewitz P, Krokowski M, Graf A, Prinz J, Puchta U, Medeiros LJ, Sander CA. Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases. Am J Pathol. 1998;152:29–33. - PMC - PubMed
    1. Greiner TC, Raffeld M, Lutz C, Dick F, Jaffe E. Analysis of T cell receptor γ gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Am J Pathol. 1995;146:46–55. - PMC - PubMed
    1. Theodorou I, Bigorgne C, Delfau MH, Lahet C, Cochet G, Vidaud M, Raphael M, Gaulard P, Farcet JP. VJ rearrangements of the TCR gamma locus in peripheral T-cell lymphomas: analysis by polymerase chain reaction and denaturing gradient gel electrophoresis. J Pathol. 1996;178:303–310. - PubMed
    1. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, Garcia-Sanz R, van Krieken JH, Droese J, Gonzalez D, Bastard C, White HE, Spaargaren M, Gonzalez M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936. Leukemia. 2003;17:2257–2231. - PubMed

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