title: T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.
abstract: We compared the effectiveness of polymerase chain reaction (PCR) and DNA blot analysis (DBA) for detecting clonal T-cell populations and investigated whether a nonradioactive PCR method could be used in routine clinical diagnosis. We analyzed DNA from 117 cases for T-cell clonality by PCR amplification. DBA was performed on 77 of these cases. Denaturing polyacrylamide gel electrophoresis (PCR-PAGE) of radiolabeled PCR products and capillary electrophoresis (PCR-CE) of fluorescently labeled PCR products were used for PCR product separation and quantitation. Complete agreement was obtained between PCR-PAGE and DBA in 67 of 77 cases. One case was positive by DBA and negative by PCR-PAGE, and 3 cases were positive by PAGE and negative by DBA. Five cases indeterminate by DBA were positive by PCR-PAGE, and 1 indeterminate case was negative by PCR-PAGE. In the comparison of PCR-PAGE and PCR-CE, of 63 cases with height ratios less than 2.0, all were negative by PCR-PAGE. Of 52 cases with height ratios of 2.0 or more, 50 were positive by PCR-PAGE. We conclude that PCR-CE is analytically equivalent to DBA and PCR-PAGE for detecting clonal T-cell populations. The PCR-CE method is semiquantitative and, therefore, may be more objective than gel-based methods.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle 98195-7110, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Sprouse
authors.forename: R
authors.initials: R
authors.lastname: Werling
authors.forename: D
authors.initials: D
authors.lastname: Hanke
authors.forename: C
authors.initials: C
authors.lastname: Lakey
authors.forename: L
authors.initials: L
authors.lastname: McDonnel
authors.forename: B L
authors.initials: BL
authors.lastname: Wood
authors.forename: D E
authors.initials: DE
authors.lastname: Sabath
mesh_terms: D002999:Clone Cells; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor / Q000235:genetics; D019675:Genes, T-Cell Receptor gamma / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets / Q000473:pathology*
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm