British Journal of Haematology

AimsTo evaluate the association between platelet indices and platelet count severity in patients with primary immune thrombocytopenia during routine post-treatment follow-up. MethodsThis retrospective observational study included patients with primary immune thrombocytopenia followed at a single tertiary care center between 2011 and 2025. Demographic and laboratory data were obtained from medical records. Platelet count severity was categorized as less than 30 x 10^9/L, 30 to 100 x 10^9/L, and greater than 100 x 10^9/L. Platelet indices, including mean platelet volume (MPV) and platelet distribution width (PDW), were analyzed using the most recent complete blood count obtained during routine follow-up after treatment initiation. Continuous variables were summarized as median and interquartile range. Comparisons across platelet count categories were performed using the Kruskal-Wallis test with post hoc Mann-Whitney U testing. Correlation analysis and simple linear regression were also conducted. ResultsA total of 243 patients were identified, of whom 232 met the inclusion criteria. Platelet distribution width differed significantly across platelet count severity categories (Kruskal-Wallis p < 0.001) and demonstrated a strong inverse association with platelet count. Mean platelet volume also showed a statistically significant difference across platelet count groups (Kruskal-Wallis p = 0.007), although the association was weaker and less consistent compared with PDW. Regression analysis confirmed a significant association between platelet count and PDW. ConclusionPlatelet distribution width is more closely associated with platelet count severity than mean platelet volume in patients with primary immune thrombocytopenia during routine post-treatment follow-up. PDW may represent a useful adjunctive laboratory parameter when interpreted alongside platelet count in routine clinical practice.

BackgroundClassical myeloproliferative neoplasms (MPN)--essential thrombocythemia, polycythemia vera, and primary myelofibrosis--are characterized by clonal hematopoiesis, overproduction of mature blood cells, and a high burden of thromboembolic events. Although thrombosis is the leading cause of morbidity and mortality in MPN, the contribution of the vascular endothelium remains incompletely defined. We investigated patient-derived endothelial colony-forming cells (ECFCs) as a surrogate for vascular endothelium in individuals with JAK2 V617F-mutated MPN. MethodsECFCs were cultured from peripheral blood of patients with MPN and healthy controls, phenotyped for thrombo-inflammatory and adhesive markers, tested for JAK2 V617F, and profiled by bulk RNA sequencing. Functional assays assessed endothelial-dependent factor Xa generation. Transcriptomes were benchmarked against public HUVEC reference datasets processed through an identical quantification pipeline. ResultsECFCs were obtained more frequently and in greater numbers from patients with MPN than from controls, indicating enhanced endothelial regenerative or activation potential. MPN ECFCs exhibited increased von Willebrand factor and P-selectin expression and release, along with elevated endothelial cell-dependent factor Xa generation, consistent with a thrombo-inflammatory, procoagulant phenotype. JAK2 V617F was not detected in any ECFC colonies, supporting a non-clonal origin of these endothelial abnormalities. Transcriptomic analysis identified 289 differentially expressed genes in MPN versus control ECFCs, with pathway enrichment revealing coordinated dysregulation of blood coagulation, platelet activation, plasminogen regulation, vascular permeability, extracellular matrix organization, and angiogenesis. Benchmarking against HUVEC datasets confirmed strong endothelial identity of ECFC-derived cells, with MPN-associated changes reflecting endothelial activation rather than loss of endothelialness. ConclusionsECFCs from patients with JAK2-mutated MPN display functional and transcriptomic signatures of endothelial dysfunction in the absence of detectable driver mutations. These findings support a model in which a primed, thrombo-inflammatory endothelium cooperates with clonal hematopoiesis to promote the heightened thrombotic risk characteristic of MPN.

Background: Large language models (LLMs) perform well on standardized medical exam questions, but their reliability for complex hematology decision making is uncertain. We compared four general-purpose LLMs (GPT-4o, GPT-o3, Claude Sonnet 4, and DeepSeek-V3) with a Virtual MDS Panel (VMP), a coordinated multi-agent AI system in which domain-specialized, rule-bound software agents (WHO/ICC guidelines; IPSS-R/IPSS-M; NCCN) collaborate to generate tumor-board-level recommendations. Methods: Each model generated diagnostic, prognostic, and treatment recommendations for 30 myelodysplastic syndrome cases. Nine international MDS experts from five institutions, blinded to model identity, completed 3,000 structured ratings using 5-point Likert scales for diagnosis, prognosis, and therapy and classified errors by severity. Results: General-purpose LLMs achieved modest expert ratings (overall mean scores: 3.7 for GPT-o3, 3.2 for GPT-4o, 3.1 for DeepSeek, and 3.0 for Claude) and contained major factual errors in at least 24% of responses. The VMP increased the proportion of outputs rated 4 or higher to 87% (vs. 34-66% for general-purpose models), improved mean scores to 4.3 overall (4.3 for diagnosis, 4.4 for prognosis, and 4.1 for therapy), and reduced major errors to 8%. Conclusions: In this blinded evaluation of 30 complex MDS cases, general-purpose LLMs produced clinically important errors at rates that raise safety concerns for autonomous hematology decision making. The VMP, a rule-bound, multi-agent architecture, approached expert-level accuracy supporting its potential role as an effective decision-support tool for MDS in the future.

BackgroundSickle Cell Anaemia (SCA), a genetic blood disorder caused by a single mutation in the beta globin gene, displays a highly variable clinical course. Hydroxyurea (HU), an effective treatment, has an unclear mechanism of action. Plasma proteins can act as biomarkers for understanding disease states and response to HU treatment in SCA patients. MethodsPlasma proteome profiling of 31 healthy individuals and 76 SCA patients, including those with and without HU treatment, was performed using a high-performance liquid chromatography system and Orbitrap mass spectrometer. Statistical analysis was performed to identify differentially abundant proteins (DAPs) between SCA patients and healthy controls. Subgroup analyses were performed to look at the impact of HU treatment on plasma proteome. ResultsOur analysis yielded 43 DAPs in the plasma of SCA patients. Global correlation and protein-protein network analysis revealed that these proteins are part of a robust interaction network. Proteins showing higher abundance (LBP, ORM1 and TFRC) were primarily associated with immune response whereas those with reduced abundance (FBLN1 and F13B) were linked to blood coagulation and proteolysis. Differential abundance of several proteins such as CD14, FCN3, LFALS3BP, LAP and TGFBI was observed in either male or female patients indicating influence of gender. Importantly, HU treatment was associated with elevated levels of haptoglobin (HP) and hemopexin (HPX), key proteins involved in free hemoglobin scavenging. Notably, DAPs such as F10, LPA, and FCN3 overlapped with proteins previously reported to be differentially abundant in beta-thalassemia patients. Moreover, multiple proteins, including APOL1, AZGP1, FBLN1, GPLD1, HPX, LGALS3BP, and TFRC correlated with clinical parameters, such as blood transfusion frequency and, vaso-occlusive crisis, and WBC and platelet counts. ConclusionsThis study identifies novel differentially abundant plasma proteins in SCA, expanding the current repertoire of disease-associated biomarkers and proteins modulated by hydroxyurea therapy. The observed overlap with beta-thalassemia associated signatures reinforces shared pathophysiological mechanisms between these hemoglobinopathies. Several of these proteins show significant correlations with key clinical parameters and disease complications, offering insights into disease mechanisms and potential utility in disease management. Collectively, these findings provide a strong foundation for translational validation in larger, independent cohorts.

Waldenstrom macroglobulinemia (WM) is a rare indolent neoplasm characterized by presence of more than 10% lymphoid cells in BM that exhibit plasmacytoid or plasma cell differentiation that secretes an IgM monoclonal protein. This is a retrospective analysis of 89 patients of WM that describes the clinical and laboratory characteristics, treatment patterns and outcome of patients of WM. The median age of the entire cophort was 66 years with male predominance (67.4%). Most common presentations were symptoms pertaining to anemia (77.5%) and constitutional symptoms (33.7%). Median bone marrow lymphoplasmacytic cells were 41%. Positivity for MYD88 and CXCR4 mutations were seen in 81.8% and 2.4% cases. BR was the most common regimen used (52.8%). Overall response rates were seen at 87.8%. Median overall survival, progression free survival and time to next treatment is 8.49 years, 2.15 years and 3.88 years. BR regimen was associated with highest event free survival.

A role for substance P in promoting neurogenic inflammation and pain has been described in sickle cell disease (SCD). However its origin and contribution to SCD pathophysiology remain unclear. We measured substance P level in plasma from 225 patients with SCD and observed the highest concentrations during acute chest syndrome (ACS). Therefore, we tested the hypothesis that substance P may induce ACS. In transgenic sickle mice, unlike control mice, intravenous injection of substance P caused lethal crises with dose-dependent acute lung injuries. Activation of Fc{varepsilon}R1 with MAR-1 had similar effects, suggesting a role for mast cell or basophil activation and degranulation. Pretreatment of sickle mice with cromolyn, a stabilizer of mast cells and basophils, prevented lethal crisis and lung injuries induced by substance P injection. In SCD patients, blood cellular histamine levels and increased histidine decarboxylase activity were consistent with an involvement of circulating basophils. Flow cytometry analysis revealed higher basophil counts with increased activation and degranulation markers in patients compared with healthy controls. During vaso-occlusive crisis, absolute basophil counts tended to decrease, suggesting their recruitment outside the vascular compartment. The same results were observed in sickle mice after hypoxia-reoxygenation, intravenous hemin injection or substance P injection. Immunohistochemistry revealed the presence of mast cells and basophils in the lungs of sickle mice, but not in control mice, with further basophil recruitment and degranulation after intravenous substance P injection. In SCD patients, we observed extremely high levels of substance P in the sputum collected during ACS, consistently with mast cell and basophil degranulation in the lungs. In vitro, substance P was shown to be a potent chemoattractant for basophils via NK1R. Gene expression analysis on sorted circulating basophils from SCD patients revealed an increased expression of several chemokine receptors, including CCR3 and FPR1, which was confirmed by spectral flow cytometry and could contribute to the recruitment of basophils in the lungs. The two substance P receptors, NK1R and MRGPRX2, were also overexpressed, promoting the vicious cycle of substance P release and pain in SCD patients. Our results reveal a novel mechanism that contributes to the understanding of ACS pathogenesis and highlights the potential role of mast cells and basophils in SCD pathophysiology.

PURPOSE: Bispecific antibodies (BsAbs) represent a major advancement in the management of relapsed/refractory multiple myeloma (RRMM), offering high response rates even in heavily pretreated patients. However, their use presents operational, safety, and supportive care complexities that require coordinated care teams, and evolving infrastructure. This manuscript summarizes best practice recommendations for adverse event (AE) management, outpatient operational models, referral pathways, and emerging strategies to optimize long-term tolerability. METHODS: Medlive, A PlatformQ Health Brand, conducted qualitative interviews of academic and community-based clinicians. Discussions focused on BsAb implementation, patient selection and counseling, and AE management. Experts provided recommendations on team-based protocols, transitions of care, and inpatient versus outpatient considerations. RESULTS: Ten hematologists/oncologists (academic n=4; community n=6) described practice patterns, barriers, and perspectives on BsAb use. BsAbs were consistently regarded as highly effective across multiple lines of therapy, particularly for patients without alternatives. Cytokine release syndrome (CRS) was the most common acute toxicity, generally low grade and managed effectively with early tocilizumab, including prophylactic use in outpatient settings. Immune effector cell-associated neurotoxicity syndrome (ICANS) was rare, mild, and best mitigated through early recognition and caregiver support. Infections, largely from BCMA-associated hypogammaglobulinemia, frequently interrupted therapy, necessitating antiviral prophylaxis, pneumocystis jirovecii pneumonia (PJP) prophylaxis, and intravenous immunoglobulin (IVIG). Outpatient step-up dosing is expanding, supported by prophylactic strategies and academic-community collaboration. Timely referral was emphasized to preserving eligibility. Major outpatient challenges included sequencing, infrastructure readiness, and standardized caregiver and staff education. CONCLUSION: Effective community implementation of BsAbs requires multidisciplinary coordination, standardized AE protocols, infection prevention, and infrastructure to support monitoring, referrals, and equitable access. These measures are critical to ensure safe, sustainable integration of bispecific therapies and to optimize patient outcomes.

ObjectivesThe therapeutic efficacy of rituximab has reduced the discriminatory power of the International Prognostic Index (IPI) in diffuse large B-cell lymphoma (DLBCL), particularly within intermediate-risk categories. To address this "risk dilution," we aimed to develop and internally validate the AB-IPI (Albumin-BCL2 Refined Prognostic Index) using a hypothesis-driven approach that integrates tumor burden, host fitness, and tumor biology. MethodsThis multi-center retrospective study analyzed 289 patients with de novo DLBCL treated uniformly with R-CHOP immunochemotherapy. We combined the standard IPI with serum albumin < 3.6 g/dL (representing host fitness/rituximab pharmacokinetics) and BCL2 protein expression > 50% (representing tumor biology). The model was validated internally using bootstrapping with 1,000 resamples in accordance with TRIPOD Type 1b guidelines. This study adhered to the TRIPOD (Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis) statement for model development and internal validation (Type 1b). ResultsDuring the observation period, 115 death events were recorded. Multivariate Cox regression identified albumin < 3.6 g/dL (Hazard Ratio 2.62), IPI score > 2 (HR 2.13), and BCL2 > 50% (HR 1.72) as independent prognostic factors. The model maintained a robust Events Per Variable (EPV) ratio of 38.3. The AB-IPI stratified patients into four distinct risk groups with 5-year overall survival rates of 88.0% (Low), 76.1% (Intermediate-1), 45.0% (Intermediate-2), and 29.0% (High). The calibration plot demonstrated excellent agreement between predicted and observed probabilities, with a calibration slope of 0.98, indicating minimal optimism and robust risk estimation. Decision Curve Analysis (DCA) demonstrated that the AB-IPI provided a superior Net Benefit across a wide range of clinically relevant threshold probabilities. ConclusionsThe AB-IPI demonstrates superior clinical utility and calibration compared to the standard IPI. By identifying patients with compounded biological risks who are unlikely to be cured by R-CHOP alone, this score offers a practical framework for optimizing therapeutic strategies, such as the allocation of polatuzumab vedotin.

ObjectivePost-thrombotic syndrome (PTS), a common complication of deep vein thrombosis, lacks objective diagnostic biomarkers and its molecular mechanisms remain poorly understood. This study aimed to identify plasma biomarkers and clarify pathways using integrated multi-omics and machine learning. MethodsProteomic and metabolomic profiling of 75 PTS patients and 75 controls was performed. Differential expression analysis, pathway enrichment, and protein-metabolite network analysis were conducted. A multi-algorithm machine learning with 8 feature selection methods prioritized biomarkers. Validations and 14 models were assessed. Results1,104 proteins and 1,891 metabolites were differentially expressed. Citrate cycle and unsaturated fatty acid biosynthesis were enriched. Three proteins, namely DIP2B, KNG1, and SUCLG2, were consistently selected as core biomarkers. All of these proteins were significantly downregulated in PTS and externally validated. A random forest model utilizing these proteins achieved an accuracy of 97.7% in independent testing, with SUCLG2 being the most influential predictor. ConclusionThis study identifies a novel three - protein biomarker panel for the diagnosis of PTS and reveals an immunometabolic axis in the pathogenesis of PTS, which links inflammatory regulation with mitochondrial energy metabolism. These findings provide valuable insights into the development of diagnostic tools and targeted therapeutic approaches.

Introduction: Solitary plasmacytomas (SP) are rare neoplasm of localised proliferation of clonal plasma cells. It can be classified based on site of involvement and bone marrow involvement. It is an indolent disease in the majority of patients. Primary modality of treatment is radiotherapy and surgical excision. Materials and methods: This was a retrospective audit of SP who were treated and followed up at a tertiary care center in eastern India from January 2012 to December 2025. Patients who has solitary plasma cytoma with more than 10% plasma cells, POEMS syndrome, have been excluded from analysis. Results: We identified 46 patients of SP. The median age of the studied population was 53 years (23-75 years). Males were more commonly affected than females (M:F=2.2:1). Most common chief complaints were bony pain (67.4%). SBP was seen in 39 (84.8%) cases whereas SEP was seen in 7 (15.2%) cases. Vertebra was the most common site of involvement (61.4%). Median M band concentration 0.24 g/dL (0.1 to 1.95 gm/dL). IgG was the most common isotype accounting for 60.6% cases. Six cases (13%) had minimal bone marrow involvement. The majority of the patients received local radiotherapy (89.1%). With a median follow up of 5.4 years (95% CI: 1.8 - 9.0), median OS was not reached, median PFS was 9.22 years (95% CI: 5.8-12.6), median time to next treatment (TTNT) was 9.86 years (95% CI: 6.8 - 12.9). Conclusion: Solitary plasmacytoma commonly affects young males. Bones are more commonly affected than extramedullary sites. SP has a lower rate of progression and excellent prognosis when treated with local radiotherapy.

Key pointsO_LIWe report findings from a phase 2 study of MY008211A among Chinese men and women aged [&ge;]18 years with paroxysmal nocturnal hemoglobinuria C_LIO_LIIncreases in hemoglobin of [&ge;]20 g/L were maintained for up to 44 weeks of treatment with MY008211A in all 34 patientsiv C_LI Explanation of noveltyParoxysmal nocturnal hemoglobinuria is characterized by red blood cell (RBC) destruction and a prothrombotic state.v Treatments exist such as complement 5 inhibitors but these carry the risk for iatrogenic extravascular hemolysis and anemia.vi As reported here, the novel, oral complement factor B inhibitor MY008211A yielded increases in hemoglobin and RBC levels, while adverse events over 44 weeks were largely mild to moderate in severity, and infections generally consisted of respiratory infections.vii Paroxysmal nocturnal hemoglobinuria (PNH) is a life-threatening disease characterized by red blood cell (RBC) destruction, blood clots, and impaired bone marrow function.viii We evaluated the efficacy and safety of 3 dosages of MY008211A, a novel complement factor B inhibitor,ix for treating PNH.x This was a multicenter, open-label, phase 2, dose-finding study of MY008211A among Chinese men and women with complement inhibitor-naive PNH and signs of active hemolysis.xi Patients with hemoglobin <100 g/L were assigned to oral MY008211A 400 mg twice daily (BID), 600 mg BID, or 800 mg once daily (QD) for 12 weeks and could then continue treatment with 400 mg BID during a 32-week extension.xii The primary endpoint was the proportion of patients achieving an increase in hemoglobin concentration of [&ge;]20 g/L vs baseline on day (D)84, without RBC transfusions after 4 weeks of dosing.xiii Safety assessments included adverse events (AEs).xiv Fifteen, 9, and 10 patients were assigned to MY008211A 400 mg BID, 600 mg BID, and 800 mg QD, respectively.xv All patients completed the study and its 32-week extension.xvi On D84, all 34 patients achieved increases in hemoglobin concentration of [&ge;]20 g/L from baseline;xvii all patients maintained this increase at D308.xviii Through D308, grade [&ge;]3 AEs occurred in 5 (33%), 5 (56%), and 4 (40%) patients in the 400-, 600-, and 800-mg groups, respectively.xix There were no deaths.xx In this multicenter, open-label study of 3 dosages of MY008211A for PNH, all patients achieved and maintained increases in hemoglobin of [&ge;]20 g/L from baseline without RBC transfusions.

Acute myeloid leukemia (AML) is a heterogeneous malignancy whose characterization relies on immunophenotyping and molecular profiling. While hemolysis is recommended for leukocyte isolation in clinical diagnostics, Ficoll-based density gradient centrifugation is widely used in research and biobanking. Here, we evaluated the impact of Ficoll isolation on commonly performed analyses of AML samples. Ficoll altered flow cytometry-based characterization by systematically enriching lymphocytes and AML blasts while depleting granulocytes. The increased T-cell content impaired AML engraftment in NSG mice, as T cells mediated terminal graft-versus-host disease. Although Ficoll had minimal impact on ex vivo AML blast expansion or chemotherapy response, RNA sequencing identified 1,136 differentially expressed genes compared with hemolysis, with Ficoll-processed samples notably leading to an overestimation of leukemic stem cell gene set expression. Immunogenomic deconvolution highlighted that Ficoll leads to an overestimation of CD8+ T-cell and monocyte abundances in sequenced samples. Mutation calling from RNA-seq data revealed substantial discrepancies between methods, including failure to detect a clinically relevant DNMT3A R882 mutation in a Ficoll-processed sample. Together, these findings support the systematic use of hemolysis to preserve cellular diversity and avoid unpredictable biases introduced by Ficoll-based isolation.

Allogeneic hematopoietic cell transplantation is the only curative option for many patients with acute myeloid leukemia (AML). In the current study, we designed and implemented a personalized assay, called v96, incorporating up to 96 mutations in 30 AML patients undergoing transplantation. The assay was performed on DNA derived in cells from the bone marrow as well as in cell-free plasma. All 30 (100%) of patients harbored molecular evidence of residual leukemia during remission that was detectable by the v96 assay, while only 6 (20%) had evidence of disease as assessed by conventional clinical assays. Furthermore, cell-free DNA from plasma proved to be more sensitive than DNA from cells of the bone marrow for identifying residual leukemia. The median number of mutants was 352-fold higher in plasma taken prior to transplantation for patients who relapsed compared to those who did not relapse. At two months post-transplantation, 27 of the 30 patients still harbored detectable leukemia as assessed by the v96 assay. Twenty-two of these patients had a subsequent decrease in leukemic burden assessed by the v96 assay, usually only after immunosuppression was discontinued and supporting a graft-versus-leukemia effect. These results document the feasibility of using a relatively large panel of carefully chosen mutations and a highly specific assay as non-invasive markers of therapeutic response in AML patients, minimizing the need for multiple bone marrow biopsies. STATEMENT OF SIGNIFICANCEWe report a blood test that tracks up to 96 patient-specific mutations and applied it to patients with AML who had undergone bone marrow transplantation. Using this test to evaluate cell-free plasma DNA, we found evidence of residual leukemia cells both during remission (prior to transplantation) in all patients, and two months following transplantation in 90% of patients. This test can mitigate the need for invasive bone marrow biopsies to follow patients with leukemia. Moreover, the test appears to be more accurate than standard assays for detecting residual leukemia, and has the potential to guide the timing of transplantation and subsequent therapeutic measures, thereby laying the foundation for future prospective studies.

iii.Background and ObjectivesThe "Pan-European Transfusion Research InfrAstructure" (PETRA) project was established to advance the use of donor, blood product, and patient datasets in Europe, aiming to benefit both patient and donor health. Here, the initial PETRA objective was to describe the landscape of existing donor and blood establishment (BE) databases. Materials and MethodsAn online survey was circulated to the European Blood Alliances BE members. The survey collected information on the feasibility of accessing donor data, and challenges and possibilities for linking these datasets with information on the associated blood products and transfusion recipients, and donors own health records. ResultsSeventeen BEs across 16 countries completed the survey. The majority could, in principle, link their donor data to product data (13 BEs (76%)) and recipient data (10 BEs (59%)), for research purposes. However, capabilities were limited and in only 29% of the BEs was the donor to recipients linkage an automated process. BEs reported significant challenges to achieve full vein-to-vein linkage, including legal constraints and lack of consent (11 BEs) and resources (10-14 BEs). IT and data issues as well as lack of knowledge and training were cited as obstacles by a minority of BEs. ConclusionWhilst the survey results suggest considerable interest in developing linkages between blood donors, their products, and recipients, many challenges remain due to a variety of obstacles. First steps in working towards a PETRA may be assistance to navigate legal frameworks as well as investing in resources and quality and harmonisation of data collections. iv. HighlightsO_LI17 blood establishments (BEs) in 16 countries responded to a survey on obstacles and opportunities for achieving vein-to-vein datasets. C_LIO_LIIn 59% of the BEs donor-to-recipient links can be established for research improving transfusion outcomes, but only in 29% this is an automated process. C_LIO_LIIn order to work towards a "Pan-European Transfusion Research InfrAstructure" (PETRA), legal frameworks, adequate donor consent and (financial and human) resources are the most common obstacles that require addressing. C_LI

Background: SARS-CoV-2 infection is an established prothrombotic trigger, yet the population-level temporal relationship between circulating viral activity and pulmonary embolism (PE) remains poorly characterized. We aimed to evaluate the short-term association between respiratory viral activity and PE hospitalizations, accounting for specific temporal lags. Methods: We conducted a population-level time-series analysis of incident PE hospitalizations in Ontario, Canada, from 2011 to 2024. Using distributed lag non-linear models, we assessed the association between standardized weekly activity levels of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) and PE risk over a 5-week lag period. Relative risks (RR) per standard deviation (SD) increase in viral activity were estimated via negative binomial regression using cross-basis terms to account for both exposure-response and lag-response non-linearities. Models were adjusted for Fourier seasonal terms and secular trends. Findings: Among 70,670 incident PE cases identified between 2011 and 2024, SARS-CoV-2 activity demonstrated a significant temporal association with PE. A cumulative RR increase of 20% per SD in SARS-CoV-2 activity was observed over the five weeks following exposure (RR 1.20; 95% CI 1.05-1.37). The risk followed a distinct delay trajectory: weekly cumulative RRs peaked at week 3 (RR 1.21; 95% CI 1.01-1.45). For the 2020-2024 period, influenza A also showed an association peaking at week 3 without statistical significance (RR 1.17; 95% CI 0.95-1.45). Interpretation: Increased population-level SARS-CoV-2 activity is associated with a heightened risk of PE, peaking at approximately the third week. This delayed peak suggests a protracted thrombo-inflammatory window, likely driven by sustained endothelial injury. These findings highlight the vascular burden of COVID-19 and suggest that infection prevention measures, including vaccination, may provide significant downstream protection against thromboembolic disease.

BackgroundCompanion canines need advances in therapeutic options for solid tumor malignancies. Prior studies established feasibility of autologous natural killer (NK) cell infusions in canines with solid tumors; however, autologous products are limited by dysfunctional immunity and a manufacturing process that delays care. Allogeneic NK cells offer the possibility of "off-the-shelf" therapy to be administered from healthy donors. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15. Additional experiments included IL-12 in the expansions. In vitro potency was assessed via co-culture with the D17-mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion. ResultsFlow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days of expansion. Residual T cell numbers varied based on donor. The addition of IL-12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic/refractory solid tumors were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well. ConclusionsCanine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro, and safety in vivo. Further studies will optimize the NK cell product and escalate dosing to reach the maximal tolerable dose.

TP53 mutations represent one of the strongest adverse prognostic factors in myelodysplastic neoplasms (MDS). While multi-hit TP53 (TP53multiHit) alterations uniformly lead to very poor outcomes, the prognostic relevance of monoallelic TP53 (TP53mono) mutations remains controversial. TP53 variants can cause loss-of-function, dominant-negative, or gain-of-function effects. We hypothesized that functional heterogeneity among TP53 variants contributes to the variable clinical behavior observed in monoallelic TP53-mutated MDS. Therefore, we analyzed pretreatment samples from 4,505 patients with MDS from two independent cohorts (IWG, n=3,173; J-MDS, n=1,332), including 271 patients with TP53mono and 499 with TP53multiHit. Functional annotation of TP53 variants was performed using a previously published phenotype score (PS) derived from saturation mutagenesis screens, capturing dominant-negative and loss-of-function effects. Median overall survival (OS) differed significantly by TP53 allelic state (TP53 wild-type (TP53wt) 42.4 months; TP53mono 22.9 months; TP53multiHit 9.2 months; p < 0.001). Within the TP53mono subgroup, functional annotation identified marked heterogeneity. Patients with high PS ([&ge;]7) showed significantly inferior OS compared with those with low PS (median OS: 13.8 vs. 39.2 months; HR 1.68, 95% CI 1.16-2.42; p = 0.006), particularly for IPSS-R and IPSS-M low-risk cases. Combining PS and variant allele frequency (VAF) further improved risk stratification. TP53mono patients with PS [&ge;]7 and VAF [&ge;]22% had outcomes comparable to TP53multiHit (median OS: 8.8, p = 0.2), whereas those with PS <7 and VAF <22% exhibited survival similar to TP53wt (median OS: 49.7, p = 0.9). Overall, functional annotation of TP53 variants refines prognostication in TP53mono-mutated MDS and may enhance individualized risk assessment.

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation. Key pointsO_LIA CROPseq-based CRISPR-Cas9 screen enables functional genomics in enucleated primary human red blood cells. C_LIO_LIChloride Intracellular Channel 3 (CLIC3) and Vesicle Associated Membrane Protein 8 (VAMP8) were identified as critical for terminal erythroid differentiation and enucleation, likely acting through two distinct mechanisms. C_LI

Understanding how specific VWF variants disrupt endothelial processing and function is central to elucidating von Willebrand disease (VWD) pathophysiology. However, current in vitro systems lack either the endothelial specificity or the genetic flexibility required for systematic variant characterization. Here, we present a genetically defined VWF-knockout cord-blood-derived endothelial colony-forming cell (VWF-KO cbECFC) model that enables controlled reintroduction of VWF variants in a physiologically relevant endothelial context. Using a patient with type 3 VWD carrying the homozygous pathogenic variant p.M771V and a second homozygous variant of uncertain significance p.R2663P as a reference, we demonstrate that expression of p.M771V in VWF-KO cbECFCs reproduces the patients intracellular processing defect and loss of high-molecular-weight multimers, whereas p.R2663P behaves as a benign allele. These findings establish the models ability to accurately distinguish pathogenic from non-pathogenic variants. Comparative analyses with HEK293 cells show that VWF-KO cbECFCs provide superior subcellular resolution, reliably forming authentic Weibel-Palade bodies (WPBs) and faithfully revealing ER retention phenotypes that remain ambiguous in non-endothelial systems. The proliferative capacity of cbECFCs further enables scalable and reproducible experimentation, overcoming major limitations associated with patient-derived ECFCs. Looking ahead, the VWF-KO cbECFC platform offers broad potential for VWF and VWD research. Its endothelial identity and genetic flexibility make it suitable for investigating VWF biosynthesis and trafficking, secretion dynamics, WPB biology, angiogenic processes, and shear-dependent VWF function. This system therefore provides a versatile foundation for mechanistic studies, systematic variant assessment, and future translational applications.

BackgroundLong COVID is characterised by persistent systemic inflammation and endothelial dysfunction, with increasing evidence implicating thromboinflammatory mechanisms. Platelet-monocyte aggregates (PMA) represent a sensitive marker of platelet activation and immune-vascular interactions, but their role in Long COVID remains incompletely defined. MethodsThis study quantified circulating PMA in 20 Long COVID patients and 20 healthy controls using a two-colour imaging flow cytometry assay targeting CD14 (a monocyte receptor for pathogen-associated molecular patterns, PAMPs) and CD62P (P-selectin). PMA were expressed as a percentage of total monocytes, and platelet attachment patterns were classified into single versus multiple platelet binding. Statistical analyses included Shapiro-Wilk normality testing, unpaired t-tests, Mann-Whitney U tests or two-way ANOVA as appropriate, and linear regression for correlation analysis. ResultsCirculating PMA were significantly elevated in Long COVID patients compared with controls (29.19 [20.02-37.26] vs 4.59 [2.67-7.16], p < 0.0001). Long COVID samples showed a reduced proportion of monocytes with single platelet attachment and a corresponding increase in multiple platelet binding (p < 0.0001). In controls, %PMA increased with age (p < 0.01), whereas no age association was observed in Long COVID, indicating an elevated baseline independent of age. ConclusionsLong COVID is associated with markedly increased platelet-monocyte aggregation and altered platelet attachment dynamics, consistent with sustained thromboinflammatory activity. PMA represent a sensitive cellular marker of platelet-driven immune activation and may have utility as an accessible biomarker for stratifying thromboinflammatory burden in Long COVID.