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JMD 2007, Vol. 9, No. 2
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Analysis of T-Cell Receptor- Gene Rearrangements Using Oligonucleotide Microchip

A Novel Approach for the Determination of T-Cell Clonality

Olga A. Gra^*, Julia V. Sidorova, Eugene A. Nikitin, Alexander Y. Turygin^*, Sergey A. Surzhikov^*, Anait L. Melikyan, Andrey B. Sudarikov, Alexander S. Zasedatelev^* and Tatyana V. Nasedkina^*

From the Department of Biological Microchips, * Engelhardt Institute of Molecular Biology, and the Department of Molecular Hematology, Hematology Research Center, Russian Academy of Medical Sciences, Moscow, Russia

T-cell clonality estimation is important for the differential diagnosis between malignant and nonmalignant T-cell proliferation. Routinely used methods include polymerase chain reaction (PCR) analysis of T-cell receptor- (TCR-) gene rearrangements followed by Genescan analysis, polyacrylamide gel electrophoresis, or heteroduplex analysis to visualize amplification products. Here, we present a new method for the analysis after PCR of TCR- rearrangements using hybridization on oligonucleotide microchip. A microchip was designed to contain specific probes for all functional variable (V) and joining (J) gene segments involved in rearrangements of the TCR- locus. Fluorescently labeled fragments of rearranged -chain from patients and donors were obtained in a multiplex nested PCR and hybridized with a microchip. The results were detected using a portable microchip analyzer. Samples from 49 patients with T-cell lymphomas or leukemias and 47 donors were analyzed for T-cell clonality by microchip and single-strand conformation polymorphism analysis, which served as a standard reference method. Comparison of two techniques showed full concordance of the results. The microchip-based approach also allowed the identification of V and J gene segments involved in the particular TCR- rearrangement. The sensitivity of the method is sufficient to determine 10% of clonal cells in the sample.