Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor γ gene rearrangements

被引:45
作者
Greiner, TC [1 ]
Rubocki, RJ [1 ]
机构
[1] Univ Nebraska, Med Ctr, Dept Pathol & Microbiol, Omaha, NE 68198 USA
关键词
D O I
10.1016/S1525-1578(10)60694-0
中图分类号
R36 [病理学];
学科分类号
100104 ;
摘要
We describe the use of fluorescent-labeled primers to analyze T-cell receptor gamma gene rearrangements (TCRgammaGR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare die performance with denaturing gradient gel electrophoresis (DGGE). in a single multiplex polymerase chain reaction (PCR) we amplified TCRgammaGR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCRgammaGR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis. protocol has 92% concordance for both TCR clonal status (23 of 25) and 95% concordance in the number of individual TCRgammaGR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCRgammaGR diluted in reactive lymphoid DNA is 2% in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCRgammaGR. The effectiveness of capillary electrophoresis is similar to DGGE.
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页码:137 / 143
页数:7
相关论文
共 37 条
[1]  
Alkan S, 2001, ARCH PATHOL LAB MED, V125, P202
[2]   Evaluation of T cell receptor testing in lymphoid neoplasms - Results of a multicenter study of 29 extracted DNA and paraffin-embedded samples [J].
Arber, DA ;
Braziel, RM ;
Bagg, A ;
Bijwaard, KE .
JOURNAL OF MOLECULAR DIAGNOSTICS, 2001, 3 (04) :133-140
[3]   High-resolution analysis of gene rearrangements in lymphoid malignancies [J].
Ayling, JF ;
Iland, HJ .
PATHOLOGY, 1999, 31 (03) :252-256
[4]   Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction [J].
Beaubier, NT ;
Hart, AP ;
Bartolo, C ;
Willman, CL ;
Viswanatha, DS .
DIAGNOSTIC MOLECULAR PATHOLOGY, 2000, 9 (03) :121-131
[5]   HETERODUPLEX ANALYSIS OF T-CELL RECEPTOR-GAMMA GENE REARRANGEMENTS FOR DIAGNOSIS AND MONITORING OF CUTANEOUS T-CELL LYMPHOMAS [J].
BOTTARO, M ;
BERTI, E ;
BIONDI, A ;
MIGONE, N ;
CROSTI, L .
BLOOD, 1994, 83 (11) :3271-3278
[6]   RAPID, NONRADIOACTIVE DETECTION OF CLONAL T-CELL RECEPTOR GENE REARRANGEMENTS IN LYMPHOID NEOPLASMS [J].
BOURGUIN, A ;
TUNG, R ;
GALILI, N ;
SKLAR, J .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (21) :8536-8540
[7]  
Chhanabhai M, 1997, AM J CLIN PATHOL, V108, P295
[8]   Rapid, multifluorescent TCRG Vγ and Jγ typing:: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations [J].
Delabesse, E ;
Burtin, ML ;
Millien, C ;
Madonik, A ;
Arnulf, B ;
Beldjord, K ;
Valensi, F ;
Macintyre, EA .
LEUKEMIA, 2000, 14 (06) :1143-1152
[9]  
Dippel E, 1999, J PATHOL, V188, P146, DOI 10.1002/(SICI)1096-9896(199906)188:2<146::AID-PATH334>3.0.CO
[10]  
2-7