Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates

Am J Pathol. 2001 Dec;159(6):2031-43. doi: 10.1016/S0002-9440(10)63055-6.

Abstract

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

MeSH terms

  • B-Lymphocytes / metabolism*
  • B-Lymphocytes / pathology
  • Base Sequence
  • Biopsy, Needle
  • Bone Marrow / pathology
  • Chromosomes, Human, Pair 11 / genetics
  • Chromosomes, Human, Pair 14 / genetics
  • Chromosomes, Human, Pair 18 / genetics
  • Clone Cells
  • DNA / chemistry
  • DNA / genetics*
  • Fixatives
  • Formaldehyde
  • Frozen Sections
  • Gene Rearrangement
  • Genes, T-Cell Receptor gamma / genetics
  • Humans
  • Immunoglobulin Heavy Chains / genetics
  • Jurkat Cells
  • Lymphoma, T-Cell / genetics
  • Lymphoma, T-Cell / pathology
  • Lymphoproliferative Disorders / blood
  • Lymphoproliferative Disorders / diagnosis
  • Lymphoproliferative Disorders / genetics
  • Molecular Sequence Data
  • Paraffin Embedding
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA
  • Sequence Homology, Nucleic Acid
  • T-Lymphocytes / metabolism*
  • T-Lymphocytes / pathology
  • Tissue Fixation
  • Translocation, Genetic*
  • Tumor Cells, Cultured

Substances

  • Fixatives
  • Immunoglobulin Heavy Chains
  • Formaldehyde
  • DNA