Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor

Abstract

High-throughput sequencing (HTS) of the T-cell receptor (TCR) has advanced rapidly in recent years, giving the unique opportunity to identify and quantify every distinct T-cell clone present within any biological sample. This sensitive and accurate technique provides the relative frequency of each individual clone within the full T-cell repertoire, allowing to study particular single T-cell clones of interest as well as the concomitant analysis of all different clones of the whole population. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or pathologic conditions, as well as to understand the development and kinetics of antigen-specific T-cell responses that critically determine protective immunity (i.e. vaccination) and immune-related disorders (i.e. auto-immunity and cancer). Interestingly, HTS can be tailored for personalized medicine, having the potential to monitor therapeutic interventions and reveal prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances and limitations of HTS, and describe emerging applications of this novel technique in the field of investigative dermatology, including studying the pathogenesis of T cells in allergic dermatitis and its application in diagnosis, early recurrence detection and therapy monitoring of cutaneous T-cell lymphoma.

Potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (COVID-19)

“…It is also worth mentioning here that virus shedding in some patients may continue for some days after symptom relief and recovery [33,34]. Notably, a very recent study showed that the communicable period (expressed as first time of SARS-CoV-2 positive to date of virus clearance) in patients infected by SARS-CoV-2 was 6 days (IQR, 2-12 days) in subjects without symptoms compared to 12 days (IQR, [12][13][14] in those who became instead symptomatic [35].…”

“…injury exposure, unreliable cold chain, prolonged transportation time), presence of interfering substances (e.g. release of cellular components that may interfere with the assay due to whole blood freezing, use of inappropriate additives) [13][14][15], as well as by a number of procedural issues occurring during sample preparation, thus including pipetting errors during manual sample preparation or aliquoting, cross-contamination and sample mismatch, among others [16]. The leading specific problems that may plague the quality of RT-PCR assays include sample contamination (even trace amounts of external DNA may jeopardize test results) and testing carried out in patients receiving antiretroviral therapy, which may then generate falsenegative results [15].…”

“…Beside these microorganism-related issues, and like other areas of diagnostic testing [40], the accuracy of RT-PCR can be substantially plagued by lack of harmonization (of primers and probes) [41], as well as by a variety of technical and analytical errors, as summarized in detail elsewhere [13][14][15]. In brief, these typically encompass instrument malfunctioning (including inappropriate PCR cycling conditions), use of insufficient or inadequate material, non-specific annealing of PCR to homologous sequences, misinterpretation of expression profiles and so forth.…”

Clonotypic Diversity of the T-cell Receptor Corroborates the Immature Precursor Origin of Cutaneous T-cell Lymphoma

“…To limit sampling error, small clusters of 10 to 20 atypical Analysis of TCRg rearrangements in microdissected mycosis fungoides samples. H&E-stained skin sections were microdissected using a laser microscope to capture small clusters of atypical "tumor" cells (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Examples of stained skin sections used for LCM are provided in Supplementary Fig.…”

Research Techniques Are Not Simple

“…I have also continued to benefit by reading the wide breadth of topics that include some privileged access to pearls of advice not shared within standard scientific publication. Examples include the comparison of different approaches to T-cell receptor analysis (Matos et al, 2017) and the practical experimental rationale to guide the choice of labeling enzymes in methods to elucidate protein interactions through spatial proximity (Che and Khavari, 2017). The bullet points of limitations to each technique hold helpful cautions and are refreshingly frank.…”