Assay design affects the interpretation of T-cell receptor gamma gene rearrangements

Comparison of the performance of a one-tube assay with the BIOMED-2-based TCRG gene clonality assay

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Abstract

Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2x ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5x peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay.

Original languageEnglish (US)
Pages (from-to)787-796
Number of pages10
JournalJournal of Molecular Diagnostics
Volume12
Issue number6
DOIs
StatePublished - Nov 1 2010

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T-Cell Receptor gamma Genes
T-Lymphocyte Gene Rearrangement
T-Cell Receptor Genes
Gene Rearrangement
Fluorescent Dyes
Color
Capillary Electrophoresis
Genes
DNA

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

Cite this

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title = "Assay design affects the interpretation of T-cell receptor gamma gene rearrangements: Comparison of the performance of a one-tube assay with the BIOMED-2-based TCRG gene clonality assay",
abstract = "Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9{\%} vs. 96.4{\%}; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5{\%} vs. 56.3{\%}; P = 0.049) when a 2x ratio was used to define clonality. Specificity was improved to 81.3{\%} by the use of a 5x peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay.",
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