Performance evaluation of the new Sysmex XR-Series haematology analyser.

Background: The new XR-Series haematology analyser from Sysmex provides increased throughput and automation, along with a new reagent in WDF channel for optimised WBC differential. Methods: An analytical performance study for the XR analyser was conducted to evaluate the WDF channel parameters in comparison to the instrument specifications. Additionally, 7460 samples were measured on XR and XN analysers to compare selected parameters and flags, and 930 randomly selected samples were further evaluated with microscopy. Results: All investigated aspects of the analytical performance study for the XR fell within the manufacturer specifications. The correlation coefficients between the two systems for the parameters tested were greater than 0.983 for the main CBC and DIFF parameters, greater than 0.909 for the Extended Inflammation Parameters, and greater than 0.932 for the parameters used in the workflow rulesets of the Extended IPU. Similarly high sensitivities for the detection of abnormal cells were observed for the 'Blasts/Abn Lympho?' flag (XN: 100%, XR: 99.0%) and WPC abnormal flags ('Blasts?' or 'Abn Lympho?') (XN: 97.0%, XR: 96.0%). XN with WPC channel had a 26% reduction of false positive smears compared to XR with 22% reduction, a statistically non-significant difference. Conclusion: The XR analyser had very good analytical performance, and highly comparable results to the predecessor XN analyser in all investigated parameters, flags and workflow aspects.

Evaluation of the Sysmex XQ-320 three-part differential haematology analyser and its flagging capabilities.

BACKGROUND: Three-part differential (3PD) haematology analysers offer a quick, easy-to-use and economical way to acquire important information about a patient's physiology. In this study, we evaluated a new 3PD analyser, the Sysmex XQ-320, investigated its comparability with its predecessor (Sysmex XP-300) and the five-part differential analyser Sysmex XN-9000, and explored its flagging potential. METHODS: Analytical performance studies were conducted for repeatability, within-laboratory precision, between-day precision, carry-over and linearity with fresh blood and QC material. Method comparison was performed in 493 samples comparing XQ-320 with XP-300, using the XN-9000 as the gold standard. RESULTS: The XQ-320 excelled manufacturer's specifications in the analytical performance studies, except for MXD in within-laboratory and between-day precisions using the QC material level 1. The XQ-320 showed correlation values greater than 0.94 with XN-9000 for the majority of the 20 reportable parameters (MXD# 0.891, MXD% 0.898 and MCHC 0.849). Improvements over the XP-300 were observed in WBC in the leucocytopenic range (bias -0.038 vs. -0.097) and PLT (bias 2.568 vs. -7.877, intercept 3.880 vs. -8.845). Concordance between XQ-320 and XP-300 was 91.9% for the WBC histogram abnormal distribution flag and 95.3% for the PLT flag. Patterns of increased neutrophils and decreased mixed cells on the XQ-320 were observed in samples that raised a flag on XN-9000. CONCLUSION: The XQ-320 showed excellent analytical performance, and very good to excellent correlation with XN-9000 with improvements over XP-300. Flagging combined with parameter patterns identified additional suspected abnormal samples, thus making the XQ-320 an excellent solution for laboratories utilising 3PD analysers.

Validation of the Sysmex XN analyser and Blood Bank mode for the quality and safety of donor blood and transfusion products.

OBJECTIVES: Our objective was to compare the measurement of residual white blood cell (rWBC) and residual red blood cell (rRBC) counts in blood products using the XN Blood Bank mode and the laboratory standard operating procedures for manual counts. In addition, to compare the whole blood complete blood count (CBC) values of blood donors and the quality of blood products using the Sysmex XN analyser versus the XS-1000i analyser. MATERIALS AND METHODS: For blood donors, 190 samples from blood or apheresis donors were analysed on both the Sysmex XS-1000i and XN-1000 analysers and the mean values of six CBC parameters were compared: the white blood cell count (WBC), the red blood cell count (RBC), haemoglobin (HGB), haematocrit (HCT), the mean corpuscular volume (MCV), the platelet count (PLT). For blood products, 164 samples were collected: 13 Plasma products - whole blood, 9 Plasma products - apheresis, 36 RBC concentrates - whole blood, 30 PLT concentrates - buffy coats, 36 PLT concentrates - buffy coats - pooled and 55 PLT concentrates - apheresis. RESULTS: All CBC parameters of the blood donors tested showed similar performance, with excellent correlation coefficients (r) ranging from 0.821 to 0.995. The majority of the blood products did not have a quantifiable number of residual cells, meaning the number of rWBC and rRBC, if present, was below the limit of quantitation (LoQ) of the different methods. rWBC were detected by Blood Bank mode in Plasma products - whole blood with a mean rWBC of 0.012 × 109 /L and in PLT concentrates - buffy coats with a mean rWBC of 0.19 × 109 /L. The correlation coefficient in both analysers for all three parameters (HGB, HCT, RBC) in RBC concentrates - whole blood was excellent, ranging from 0.95 to 0.99. For platelet count, r ranged from 0.98 to 0.99. CONCLUSION: The XN-Series analyser, equipped with a Blood Bank mode, demonstrated reliable performance when used for blood donor evaluation, rWBC enumeration and measurement of end blood products.

Posttransplantation MRD monitoring in patients with AML by next-generation sequencing using DTA and non-DTA mutations.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) is widely applicable and prognostic prior to allogeneic hematopoietic cell transplantation (alloHCT). We evaluated the prognostic role of clonal hematopoiesis-associated DNMT3A, TET2, and ASXL1 (DTA) and non-DTA mutations for MRD monitoring post-alloHCT to refine MRD marker selection. Of 154 patients with AML, 138 (90%) had at least one mutation at diagnosis, which were retrospectively monitored by amplicon-based error-corrected NGS on day 90 and/or day 180 post-alloHCT. MRD was detected in 34 patients on day 90 and/or day 180 (25%). The rate of MRD positivity was similar when DTA and non-DTA mutations were considered separately (17.6% vs 19.8%). DTA mutations had no prognostic impact on cumulative incidence of relapse, relapse-free survival, or overall survival in our study and were removed from further analysis. In the remaining 131 patients with at least 1 non-DTA mutation, clinical and transplantation-associated characteristics were similarly distributed between MRD-positive and MRD-negative patients. In multivariate analysis, MRD positivity was an independent adverse predictor of cumulative incidence of relapse, relapse-free survival, and overall survival but not of nonrelapse mortality. The prognostic effect was independent of different cutoffs (above limit of detection, 0.1% and 1% variant allele frequency). MRD log-reduction between diagnosis and post-alloHCT assessment had no prognostic value. MRD status post-alloHCT had the strongest impact in patients who were MRD positive prior to alloHCT. In conclusion, non-DTA mutations are prognostic NGS-MRD markers post-alloHCT, whereas the prognostic role of DTA mutations in the posttransplant setting remains open.

Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia.

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

Emerging strategies to target the dysfunctional cohesin complex in cancer.

INTRODUCTION: Mutations in cohesin genes have been described in numerous solid cancers and hematologic malignancies; subsequent experimental evidence has linked these mutations with carcinogenesis. Areas covered: In this review, we present current information about the physiological role of the cohesin complex in normal and malignant cells and describe current therapeutic strategies that are being explored in cohesin-mutated cancers. We discuss a range of targets and strategies that should be explored to develop targeted therapies for patients with aberrant cohesin. Expert opinion: Targeting of the cohesin complex is an underexplored area of drug development. There is a high frequency of cohesin mutations in multiple cancers, hence specific targeting strategies should be explored. Cohesins play a crucial role in cellular organization; therefore, we expect a narrow therapeutic window of direct inhibitors of cohesin components. Exploiting experimental approaches that correct dysfunctional cohesins and coupling them with current therapeutic strategies can provide novel, innovative and more effective treatment regimens.

Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress antitumor immune responses, promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of antitumor immunity. Specifically, MDSCs in patients with melanoma and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells markedly delayed tumor growth and endowed antitumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, whereas transcriptome analysis revealed substantial differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation, thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase that mediates the lysosomal degradation of MHC II in M-MDSCs attenuated their suppressive function, and resulted in markedly decreased tumor volume followed by development of a robust antitumor immunity. Collectively, these findings depict autophagy as a molecular target of MDSC-mediated suppression of antitumor immunity.

Elimination of Granulocytic Myeloid-Derived Suppressor Cells in Lupus-Prone Mice Linked to Reactive Oxygen Species-Dependent Extracellular Trap Formation.

OBJECTIVE: Emerging evidence supports a crucial role of myeloid-derived suppressor cells (MDSCs) in the regulation of autoimmune diseases. However, their role in systemic lupus erythematosus (SLE) remains unknown. This study sought to address the role of MDSCs in the pathogenesis of SLE. METHODS: MDSCs from (NZB × NZW)F1 lupus-prone mice were assessed for phenotype by flow cytometry, and the function of MDSCs was analyzed by in vitro T cell proliferation assay and real-time quantitative polymerase chain reaction. Extracellular trap (ET) formation was evaluated by immunofluorescence and confocal microscopy. The production of reactive oxygen species (ROS) by Ly-6G+ cells was determined by fluorescence-activated cell sorting analysis. RESULTS: Expansion of MDSCs was impaired and the function of MDSCs was defective in the lymphoid organs of (NZB × NZW)F1 lupus-prone mice with established disease, in which involvement of predominantly the granulocytic MDSC (G-MDSC) cell subset was observed. More specifically, the results showed that increased elimination of G-MDSCs, driven by the inflammatory milieu of lupus, could be attributed to ET formation, and that cytokines, such as interferon-α (IFNα), IFNγ, and interleukin-6, play a role in this process. Induction of ET release by G-MDSCs was mediated by the production of ROS, since inhibition of ROS generation significantly reduced ET release. CONCLUSION: Collectively, the results of this study reveal that elimination of a crucial regulatory immune cell subset is a feature of the SLE microenvironment. These findings provide new insights into the pathogenetic mechanisms of the disease.