Analysis of T-cell clonality using laser capture microdissection and high-resolution microcapillary electrophoresis

Identification of clonal lymphocytic populations by polymerase chain reaction may be difficult in cases with scant cellular infiltrates or those with a heterogeneous population of cells. Here, we assessed the diagnostic utility of laser capture microdissection (LCM) and high-resolution microcapillary electrophoresis in the analysis of clonality of small biopsy specimens. Clonality was determined in 24 cases: five reactive tonsils, five reactive lymph nodes, six inflammatory skin lesions, and eight T-cell lymphomas. CD3-positive T lymphocytes were captured by LCM from paraffinized immunohistochemically stained sections. Genomic DNA was analyzed for T-cell receptor-gamma gene rearrangement by polymerase chain reaction followed by high-resolution microcapillary electrophoresis with the DNA 500 LabChip and the Agilent Bioanalyzer. In the reactive specimens, T-cell receptor-gamma polymerase chain reaction revealed monoclonal bands when 10 to 1000 cells were captured. This pattern changed to polyclonal when higher numbers of cells were microdissected (2000 to 10,000 cells). In contrast, lymphoma cells were consistently monoclonal whether low or high numbers were microdissected. Microcapillary electrophoresis coupled with LCM facilitated clonality analysis in equivocal cases. In two of eight lymphoma cases, LCM revealed diagnostic monoclonal bands, whereas routine T-cell receptor-gamma assessment of whole tissue sections with 10% polyacrylamide gel electrophoresis demonstrated only minor clonal bands. We conclude that clonality determined by LCM is cell number-dependent. Biopsy specimens containing low numbers of reactive polyclonal T cells may produce pseudomonoclonal bands and therefore should be interpreted with great caution.