Pitfalls in TCR gene clonality testing: teaching cases

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis

Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (approximate to transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 degrees C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for < 24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population

Improved standardization of flow cytometry diagnostic screening of primary immunodeficiency by software-based automated gating

Background Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt (TM) software (Cytognos). For comparison, percentage differences in absolute cell counts/mu L were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/mu L) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles

Highly sensitive MRD tests for ALL based on the IKZF1 Δ3–6 microdeletion

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IgCaller for reconstructing immunoglobulin gene rearrangements and oncogenic translocations from whole-genome sequencing in lymphoid neoplasms

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.We are indebted to the Genomics Core Facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for the technical support, to R. Siebert and D. Huebschmann for sharing the CSR regions, and to K. Stamatopoulos, E. Vlachonikola and F. Psomopoulos for their helpful comments on the manuscript. We thank R. Eils, P. Lichter, C. von Kalle, S. Fröhling, H. Glimm, M. Zapatka, S. Wolf, K. Beck, and J. Kirchhof for infrastructure and pipeline development within DKFZ-HIPO and NCT POP. This study was supported by the Instituto de Salud Carlos III and the European Regional Development Fund “Una manera de hacer Europa” (PMP15/00007 to E.C.), the “la Caixa” Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221 to E.C.), the National Institute of Health “Molecular Diagnosis, Prognosis, and Therapeutic Targets in Mantle Cell Lymphoma” (P01CA229100 to E.C.), and CERCA Programme/Generalitat de Catalunya. F.N. is supported by a pre-doctoral fellowship of the Ministerio de Economía y Competitividad (BES-2016-076372). F.M. is supported by the Memorial Sloan Kettering Cancer Center NCI Core Grant (P30 CA 008748). E.C. is an Academia Researcher of the “Institució Catalana de Recerca i Estudis Avançats” (ICREA) of the Generalitat de Catalunya. This work was partially developed at the Centre Esther Koplowitz (CEK, Barcelona, Spain).Peer ReviewedPostprint (published version

Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings