RESUMEN
Mature T-cell lymphomas consisting of an expanded clonal population of T cells that possess common rearrangements of the T-cell receptor (TCR) encoding genes can be identified and monitored using molecular methods of T-cell repertoire analysis. We have developed a hybrid-capture method that enriches DNA sequencing libraries for fragments encoding rearranged TCR genes from all 4 loci in a single reaction. We use this method to describe the TCR repertoires of 63 putative lymphoma clinical isolates, 7 peripheral blood mononuclear cell (PBMC) populations, and a collection of tumor infiltrating lymphocytes. Dominant Variable (V) and Joining (J) gene pair rearrangements in cancer cells were confirmed by polymerase chain reaction (PCR) amplification and Sanger sequencing; clonality assessment of clinical isolates using BIOMED-2 methods showed agreement for 73% and 77% of samples at the ß and γ loci, respectively, whereas ß locus V and J allele prevalence in PBMCs were well correlated with results from commercial PCR-based DNA sequencing assays (r 2 = 0.94 with Adaptive ImmunoSEQ, 0.77-0.83 with Invivoscribe LymphoTrack TRB Assay). CapTCR-seq allows for rapid, high-throughput and flexible characterization of dominant clones within TCR repertoire that will facilitate quantitative analysis of patient samples and enhance sensitivity of tumor surveillance over time.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Análisis de Secuencia de ADN/métodos , Biblioteca de Genes , Reordenamiento Génico de Linfocito T/genética , Sitios Genéticos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Linfoma de Células T/diagnóstico , Linfoma de Células T/genética , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/química , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismoRESUMEN
CONTEXT: Many hematology laboratories have adopted semi-automated digital platforms for routine use and the evidence supporting their use is increasing. AIMS: The CellaVision platforms are among the most thoroughly studied digital hematology platforms; we wished to determine the accuracy of CellaVision for reticulocyte counting. DESIGN MATERIALS AND METHODS: We compared reticulocyte counts performed manually, using the Beckman Coulter LH750 automated analyzer and with the CellaVision DM96 platform. We analyzed the results for pair-wise correlation and bias, and precision. STATISTICAL ANALYSES USED: Analyses were performed using Statistical Package for the Social Sciences software (SPSS), including Spearman's rho correlation coefficient, Friedman's two-way Analysis Of Variance (ANOVA) for comparison of distributions; bias was compared by way of mean and standard deviation. RESULTS: The CellaVision reticulocyte counts correlated most strongly with those of the analyzer (often considered the benchmark test); the reticulocyte count distributions were noted not to be significantly different from each other across all three methods. The mean and standard deviation of bias were lowest in the comparison of CellaVision and LH750 counts. CONCLUSIONS: Our data provide additional support for the accuracy of digital hematology applications using the CellaVision DM96 platform.