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Unit Code 83122:
T-Cell Receptor Gene Rearrangement

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Method Description

PCR analysis will be performed on all specimens. Southern blot

analysis will be performed on most cases with sufficient DNA

available, unless such testing is not deemed necessary after

correlation with other clinicopathologic information.

 

In the PCR assay, the TCR gamma (TCRy) gene is analyzed because

it is rearranged early in the development of all T-cells. Genomic DNA

is extracted from the specimen. PCR primers are used that were

designed to amplify a fragment that spans the junctions of the variable

and joining segments and should amplify the majority of all possible

rearrangements (rearrangements containing the yJ1.2 exon will not

be detected by the assay). Four primers to variable segments and 2

primers to joining segments are used in a single reaction. Each unique

rearrangement produces a PCR fragment of a unique size. The upstream

primers are labeled with a fluorescent "tag" so that the PCR product

can be detected. The PCR fragments are analyzed by capillary gel

electrophoresis using the Applied Biosystems (ABI) 3100 machine

for fragment size and amount.

 

In the Southern blot assay, genomic DNA is digested with the restriction

enzyme Eco R1. The fragments are separated using agarose gel

electrophoresis, transferred to a nylon membrane and hybridized with

probes labeled with alkaline phosphatase. Two probes (1 to the

first TCR beta gene joining region and 1 to the second TCR beta

gene joining region) are used on separate membranes. Fragments

are detected using a chemiluminescent-based system: alkaline

phosphatase bound to the probe catalyzes the decomposition of a

dioxetane substrate (CPD-Star) to produce light that is detected by

radiographic film. (Greiner TC, Raffeld M, Lutz C, et al:  Analysis of T-cell

receptor-gamma gene rearrangements by denaturing gradient gel

electrophoresis of GC-clamped polymerase chain reaction products.

Correlation with tumor-specific sequences. Am J Pathol 1995 Jan;

146(1):46-55; McCarthy KP, Sloane JP, Kabarowski JH, et al:  A simplified

method of detection of clonal rearrangements of the T-cell receptor-gamma

chain gene. Diagn Mol Pathol 1992 Sep;1(3):173-179; Medeiros LJ,

Weiss LM:  The utility of the polymerase chain reaction as a screening

method for the detection of antigen receptor gene rearrangements.

Hum Pathol 1994 Dec;25(12):1261-1263; Trainor KJ, Brisco MJ, Wan JH,

et al:  Gene rearrangement in B- and T-lymphoproliferative disease detected

by the polymerase chain reaction. Blood 1991 Jul 1;78(1):192-196; American

Biosciences UK Limited. 2004. Gene Images AlkPhos Direct Labeling and

Detection system [probe labeling kit instruction manual])

Performing Laboratory Location

Rochester

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