A New and Simple TRG Multiplex PCR Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium

T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffinembedded (FFPE) tissue samples. Their differential performance was mainly explained by (i)

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

Most consensus leukemia lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies