A novel method for interpretation of T-cell receptor gamma gene rearrangement assay by capillary gel electrophoresis based on normal distribution.
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.
Kuo
Frank C
FC
Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA. fkuo@partners.org
Hall
Dimity
D
Longtine
Janina A
JA
D000465:Algorithms; D015139:Blotting, Southern; D019075:Electrophoresis, Capillary / Q000379:methods*; D005069:Evaluation Studies as Topic; D015321:Gene Rearrangement / Q000235:genetics*; D019675:Genes, T-Cell Receptor gamma / Q000235:genetics*; D006801:Humans; D016399:Lymphoma, T-Cell / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*