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JMD 2007, Vol. 9, No. 1
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Technical Advance

A Novel Method for Interpretation of T-Cell Receptor Gene Rearrangement Assay by Capillary Gel Electrophoresis Based on Normal Distribution

From the Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts

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 J and four sets of V 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 ² error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for V_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.