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Effectiveness of Capillary Electrophoresis Using Fluorescent-Labeled Primers in Detecting T-Cell Receptor γ Gene Rearrangements Timothy C. Greiner, Ronald J. Rubocki The Journal of Molecular Diagnostics Volume 4, Issue 3, Pages (August 2002) DOI: /S (10) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 1 Schematic of gene segments in the T-cell receptor γ gene. V, Variable region; J, Joining region; GP, Group; C1, Constant region. The Journal of Molecular Diagnostics 2002 4, DOI: ( /S (10) ) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 2 The height (h2) of the peak must exceed the height (h1) of the polyclonal background by a ratio of more than 2.0 (RPB = h2/h1) to define a clonal result. Evaluation of bialellic TCRγGR in cell lines where one peak (h3) is outside the normal distribution of polyclonal T cells suggests that the ratio of such peaks (h3) are better assessed by adding h3 to h1 to calculate h2. Otherwise one peak will be called positive and one peak will be called negative. The Journal of Molecular Diagnostics 2002 4, DOI: ( /S (10) ) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 3 A: Polyclonal TCRγ gene rearrangements are demonstrated by the normal distribution pattern obtained from peripheral blood lymphocytes. Labeling of the TCRγGRs is as follows: the common joining region genes Jγ1/Jγ2-black, JγP-blue, and JγP1/JγP2-green. B: Molt4 cell line with biallelic Vγ2-JγP1/JγP2, Vγ10-JγP1/JγP2 TCRγGR. Molt4 DNA was diluted 50:50 in peripheral blood lymphocyte DNA before PCR. The green peaks identify that the JγP1/JγP2 joining segment is used in both TCRγGR. GR = gene rearrangement. Peak height of a clonal peak should be a minimum of two times the height of the polyclonal background. C: Fluorescent analysis of dual TCRγGR (Vγ2-JγP and Vγ9-JγP1/JγP2) in a peripheral T-cell lymphoma, demonstrating the capability of identifying different joining region genes used in each rearrangement. JγP-blue; JγP1/JγP2-green. D: Sensitivity study by serial dilution of TCRγGR (Vγ3-Jγ1/Jγ2; Vγ4-Jγ1/Jγ2) CEM in peripheral blood lymphocytes. A detection sensitivity of at least 0.5% was obtained with the CEM cell line DNA serially diluted in peripheral blood lymphocyte DNA. Below 0.5%, the ratio (RPB) of the peak height of the TCRγGRs will fall below two times of the polyclonal background. E: Discriminating a clonal peak (right arrow) from the background polyclonal T-cells in this case is difficult. Neither of the two peak heights (left arrows) is greater than two times the maximum height (at 1200) of the main polyclonal background. This illustrates the potential problem that can occur with identifying the significance of oligoclonal peaks. F: Polyclonal TCRγ gene rearrangements are demonstrated in peripheral blood lymphocytes using variable region primers labeled as follows: the common variable region genes of Vγ2–8 and Vγ3-black. The lesser common genes are Vγ9-green; Vγ10-red; Vγ11-blue. G: Fluorescent analysis identifies the variable gene used in dual TCRγGR (Vγ2 and Vγ10). in a peripheral T-cell lymphoma., Vγ2-black; Vγ10-red. x axis, nucleotide length; y axis, intensity of signal; Rox-labeled size standard at 200 nt. The Journal of Molecular Diagnostics 2002 4, DOI: ( /S (10) ) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
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