BACKGROUND:JAK2 V617F variant allele frequency (VAF) reflects clonal burden in myeloproliferative neoplasms, but the clinical associations of low-level VAF in patients with erythrocytosis remain uncertain. OBJECTIVE:The objective of this study is to examine associations between diagnostic JAK2 V617F VAF categories and clinical, laboratory, bone marrow and thrombotic outcomes in patients with JAK2 V617F-positive erythrocytosis. PATIENTS AND METHODS:This single-centre retrospective cohort included 174 adults. Patients were stratified by diagnostic VAF as low (< 5%; n = 62), intermediate (5%-50%; n = 81) or high (> 50%; n = 31). Clinical and laboratory characteristics, bone marrow findings and incident thrombotic events during follow-up were compared. Multivariable logistic regression was used to evaluate factors associated with thrombosis. RESULTS:Follow-up duration was comparable amongst the VAF groups (median, 5.0 vs. 8.0 vs. 6.0 years; p = 0.227). Thrombosis rates increased across the groups (12.9% vs. 22.2% vs. 48.4%; p = 0.001), as did splenomegaly (25.8% vs. 53.1% vs. 64.5%; p < 0.001) and myelofibrosis transformation (1.6% vs. 13.6% vs. 20.0%; p = 0.011). Amongst patients with available measurements, median serum erythropoietin decreased with higher VAF (8.0 vs. 3.1 vs. 2.2 mIU/mL; p < 0.001), whereas the proportion with subnormal erythropoietin increased (17.8% vs. 47.6% vs. 82.6%; p < 0.001). Amongst biopsied patients, polycythemia vera-supportive bone marrow morphology was present in 50.0%, 93.5% and 100% of the groups respectively. A diagnostic VAF > 50% was independently associated with subsequent thrombosis (OR: 3.35; 95% CI: 1.06-10.58; p = 0.039), as was platelet count (OR: 9.92; 95% CI: 1.52-64.63; p = 0.016). CONCLUSIONS:A diagnostic JAK2 V617F VAF > 50% was independently associated with subsequent thrombosis in this cohort. The low-VAF group was diagnostically and clinically heterogeneous. These findings should be considered hypothesis-generating and require prospective validation.
Examination of the bone marrow (BM) remains fundamental to the diagnosis, classification, prognostication, and monitoring of hematolymphoid and other disorders affecting blood cell production. Since publication of the International Council for Standardization in Haematology (ICSH) guideline in 2008, advances in diagnostic technologies, disease classification systems and laboratory reporting practices have significantly evolved. These developments necessitate a framework to support contemporary BM evaluation and reporting. This document, prepared by an international ICSH Working Party, provides updated guidance on the examination and reporting of BM specimens and is an extension and refinement of the 2008 ICSH guidelines. It outlines the appropriate application and integration of key diagnostic modalities, including morphological assessment of BM aspirate and trephine biopsy specimens, immunophenotypic analysis by flow cytometry and immunohistochemistry and molecular genetic testing. Central to this guidance is the principle of integrated reporting, whereby findings from all diagnostic modalities are synthesized within the clinical context and aligned with current international classification systems. The ICSH acknowledges the emergence of automated digital microscopy, computational decision support tools, and other technological innovations in BM analysis. These developments hold considerable promise, but whilst many remain under evaluation, specific guidance regarding their routine clinical implementation is beyond the scope of this document.
INTRODUCTION:It is recommended to detect lupus anticoagulant (LA) using two phospholipid-dependent clotting assays, including a LA-sensitive activated partial-thromboplastin-time (aPTT). Among these reagents, Staclot-LA assay is performed on a mixture of test and normal plasma with and without hexagonal phase phospholipids. However, its lack of validation on automated coagulation analyser is a major drawback. We aimed i/to assess the analytical performance of Staclot-LA assay on automated system; ii/to compare inter-laboratory agreement of Staclot-LA performed on semi-automated or fully automated analysers using two external-quality-assurance (EQA) programs data. METHODS:We adapted the Staclot-LA assay (Stago) set-up on a STA-R-Max analyser and locally validated the manufacturer's recommended cut-off for positive LA testing using samples from healthy volunteers. Reproducibility and accuracy were assessed using internal quality controls (IQC, Staclot-LA1/LA2) over a five-year period. Staclot-LA results from EQA Probioqual and ECAT were compared between semi- and fully-automated analysers. RESULTS:Agreement of our LA results (positive or negative) was observed in 100% of IQC (n = 130) and EQA (n = 58) samples. Probioqual and ECAT interlaboratory Staclot-LA results showed full agreement for 96.2% and 89.7% in semi-automated system users vs 96.7% and 92.5% in automated system users. The system had no significant impact on the qualitative results (Probioqual p = 1.000; ECAT p = 0.5912), even though we observed increasing biases of the differences in clotting times, especially in LA positive samples on automated vs semi-automated analysers. CONCLUSION:Staclot-LA assay can be reliably performed on a fully automated system, thus paving the way for wider accessibility and widespread use.
The absence of Duffy antigen expression on red blood cells, caused by a single nucleotide polymorphism in the DARC/ACKR1 gene, confers protection against malaria and is associated with lower absolute neutrophil counts (ANC) in some studied populations. This study aimed to establish reference intervals for ANC and evaluate other hematologic parameters influenced by Duffy status in Brazilian individuals. In this prospective study, complete blood counts and Duffy phenotyping were performed in 311 healthy adult blood donors from two independent blood banks. Among participants, 51.4% were male, with a median age of 37 years, and 46.6% self-identified as Black or mixed-race. The Duffy null phenotype was observed in 146 individuals (46.9%), with lower male representation and higher prevalence among Black individuals. Duffy null individuals showed lower median white blood cell counts (5350 vs. 7060/μL) and ANC (median 2250 vs. 4070/μL), with a 95% reference range of 1180-4800/μL versus 2200-7250/μL. They also had a lower neutrophil percentage and higher relative counts of other leukocytes, without differences in absolute lymphocytes and eosinophils. Additionally, they exhibited higher erythrocyte counts, hemoglobin, hematocrit, platelet counts, and mean platelet volume. These findings support Duffy-specific reference intervals to avoid misclassification in diverse populations like Brazil.
The morphological analysis of peripheral blood cells is increasingly adopting deep learning, mainly for the automatic recognition of the variety of normal and abnormal cell classes. However, its clinical application is limited by the scarcity of annotated datasets for rare diseases and the high variability of staining protocols between laboratories. This paper provides an overview of how Generative Artificial Intelligence methods can help overcome these obstacles. Firsts, some basic concepts are presented, distinguishing between discriminative AI and generative AI, specifically generative adversarial networks (GANs) and diffusion models. The core of the paper addresses two problems: (1) the automatic generation of artificial blood cell images and (2) the digital artificial staining to reduce inter-laboratory differences. In these two cases, three sections are included: underlying concepts, a literature review and practical examples. These show how high-quality images of blood cells with realistic morphological characteristics are created, which strengthen the classifier's training. In multicenter tests, the examples illustrate that normalizing the staining allows a classifier trained on abnormal blood cell images from a single hospital to accurately recognize abnormal cells obtained in other laboratories, significantly improving performance without distorting cell morphology. A final section concludes the paper with observations and future perspectives on how generative tools can assist clinical pathologists as decision support systems that can operate consistently across diverse clinical settings.
INTRODUCTION:Digital morphology (DM) analyzers may improve laboratory efficiency; however, their practical integration into manual slide review (MSR) criteria and the resulting impact on overall efficiency remain underexplored. We evaluated the efficiency of the DI-60 (Sysmex, Kobe, Japan) by assessing its potential to modify or replace existing MSR criteria in a routine practice and comparing its turnaround time (TAT) with that of manual counting. METHODS:A total of 907 samples were consecutively selected based on our laboratory's MSR criteria. We simulated modified MSR criteria incorporating the DI-60 to assess the potential reduction in laboratory workload. We compared TATs of the DI-60 and manual counting according to white blood cell (WBC) counts (range, 0.01-158.6 × 109/L). RESULTS:DI-60 achieved complete analysis in 717/907 samples (79.1%), showing stable performance in samples with normal WBC counts. However, the overall TAT of the DI-60 was significantly longer than that of manual counting (263 vs. 120 s, p < 0.001). Incomplete analysis (< 200-cell count) occurred in 190 samples (20.9%), predominantly in hematology-oncology samples (94.7%) and samples with severe leukopenia. Simulation of modified MSR criteria incorporating the DI-60 demonstrated a potential workload reduction of 11.6% (105/907). CONCLUSIONS:DM analyzers may have the potential to reduce laboratory workload by being incorporated into each laboratory's MSR criteria. With incomplete analyses in specific sample types, however, DM analyzers cannot fully replace MSR. The effective use of DM analyzers requires streamlined integration into the existing laboratory workflow and further optimization considering each laboratory's situation and unmet needs.
BACKGROUND:Flow cytometry and real-time quantitative polymerase chain reaction (qRT-PCR) for MYD88 mutations (MYD88mt) are routinely performed on bone marrow aspirates during the diagnostic evaluation of IgM monoclonal paraproteinemia. The potential predictive value of immunophenotypic findings for molecular results, however, remains to be fully clarified. OBJECTIVE:This study aimed to determine whether the presence or absence of clonal B-cells detected by flow cytometry in bone marrow aspirates can predict MYD88mt status as assessed by qRT-PCR. METHODS:A total of 88 cases with IgM monoclonal paraprotein and available bone marrow immunophenotyping and MYD88 mut qRT-PCR results were retrospectively analyzed. Flow cytometry was used to identify clonal B-cell populations, and results were correlated with molecular findings. RESULTS:The absence of clonal B-cells by flow cytometry demonstrated a high negative predictive value (NPV) of 96% for MYD88mt. Conversely, the presence of a clonal B-cell population showed a positive predictive value (PPV) of 62% for detecting MYD88mt by molecular testing. CONCLUSIONS:Bone marrow immunophenotyping by flow cytometry may serve as an effective screening tool to stratify patients with IgM monoclonal paraproteinemia for MYD88mt molecular testing. Specifically, the absence of clonal B-cells strongly predicts a negative molecular result, potentially reducing unnecessary molecular analyses.
INTRODUCTION:Bivalirudin is an alternative anticoagulant to heparin and is widely used in pediatric extracorporeal life support (ECLS). Its activity is monitored using activated partial thromboplastin time (aPTT) and/or more specific tests such as plasma-diluted thrombin time (dTT). A new automated thrombin generation assay (TGA) has also shown promise in evaluating the hemostatic response in anticoagulated patients; however, its utility for monitoring bivalirudin or other anticoagulants in pediatrics remains unknown. We aimed to evaluate TGA in pediatric patients treated with bivalirudin and to compare it with that observed in patients receiving other commonly used anticoagulants. METHODS:TGA, aPTT, aPTT with hepzyme, dTT, INR were measured in 30 specimens from 12 pediatric ECLS patients anticoagulated with bivalirudin. In addition, TGA, anti-Xa assay, and INR were measured in samples from children receiving enoxaparin (n = 22, 10 patients), heparin (n = 16, 8 patients), and warfarin (n = 19, 6 patients). RESULTS:TGA lag ratio showed a strong correlation with bivalirudin concentration as measured by dTT and ROC analysis demonstrated that a Lag ratio > 2.0 predicted specimens within the therapeutic range for bivalirudin (sensitivity 0.88, specificity 0.67, accuracy 0.79). Interestingly, samples containing endogenous heparin-like substances exhibited paradoxically enhanced thrombin generation, despite having the highest aPTT values. Enoxaparin effect was associated with a reduction in thrombin peak and endogenous thrombin potential (ETP) suppression, while both heparin and warfarin led to a prolonged lag time, along with decreased peak and greater ETP suppression. CONCLUSION:TGA shows promise in evaluating bivalirudin anticoagulation and is capable of detecting anticoagulant activity across both subtherapeutic and therapeutic levels of other agents commonly used in pediatric patients.
INTRODUCTION:An altered T-cell repertoire with aberrant immune activation is central to the pathogenesis of acquired aplastic anemia (aAA), and miRNAs are known to orchestrate T-cell activation; their relationship is, however, incompletely explored in aAA. The study aimed to validate specific miRNA-mRNA pairs involved in aberrant T-cell activation, differentiation, and cytokine levels in aAA. METHODS:This was a prospective case-control study of 30 newly diagnosed patients with aAA and 10 healthy controls. The alterations in the expression of seven miRNAs were evaluated in the sorted T-cells isolated from the PB-MNCs. qRT-PCR was used to evaluate the miRNA expression, and fold change was calculated by the 2ΔΔCt method. Target genes of the deregulated miRNAs were assessed by qRT-PCR. Cytokine profiling was carried out by cytokine bead array and qRT-PCR. RESULTS:All seven miRNAs were differentially expressed in aAA, miR-155 (FC: 3.71) and miR-146b (fold change (FC): 5.86) were upregulated, while miR-126 (FC: 0.305), miR-181c (FC: 0.343), miR-16 (FC: 0.351), miR-326 (FC: 0.219), and miR-17 (FC: 0.38) were downregulated. These alterations correlated with disease severity. Target analysis showed increased expression of transcription factors promoting Th1/Th17 polarization and signaling molecules (PIK3R2, SOCS-1), with reduced expression of FOXP3, IRAK-1, TRAF-6, STAT-1, IFNGR, ETS-1, and PTEN. The miRNA-mRNA alterations in enriched T-cells mirrored those in PB-MNCs except for miR-17. Plasma levels of IL-2, IL-4, IL-6, and IL-10 (p = 0.069) were significantly elevated in aAA. CONCLUSION:This study confirms the differential expression of miRNAs in aAA and their possible role in maintaining the aberrant T-cell repertoire in aAA. In vitro studies utilizing miRNA knockdown/miR-mimics may uncover newer therapeutic targets in aAA.
INTRODUCTION:Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy with variable clinical outcomes. Epigenetic alterations, particularly DNA methylation, have emerged as key modulators of gene expression and disease progression. This study investigates the prognostic relevance of methylation and expression profiles of CEBPA and DNMT3A in AML. METHODS:A cohort of adult AML patients from the TCGA-LAML dataset was analyzed for gene expression and methylation profiles across seven AML-associated genes. Survival associations were evaluated using Kaplan-Meier curves and Cox proportional hazards models. An initial panel of seven AML-associated genes (CEBPA, DNMT3A, HDAC1, IDH1, IDH2, NPM1, and RUNX1) was screened for prognostic relevance using matched expression and methylation data; genes with significant survival associations were retained for model construction. Patients were stratified into low, intermediate, and high-risk groups based on this score. RESULTS:Significant survival associations were observed for DNMT3A expression (p = 0.0062) and CEBPA methylation (p = 0.0074). Of the seven candidates, only DNMT3A expression and CEBPA methylation demonstrated independent prognostic value and were integrated into the final composite score. The composite score yielded strong survival separation across risk groups (p = 0.0033). In the final multivariate Cox model (n = 158), CEBPA methylation and DNMT3A expression remained independently predictive of overall survival. CONCLUSION:Methylation and expression profiles of CEBPA and DNMT3A could be integrated into a composite molecular score to complement conventional genetic and cytogenetic classifications to stratify survival risk in AML; however, further studies with established clinical risk systems are required.
INTRODUCTION:Complete blood count (CBC) discrimination indices are widely used as low-cost triage tools for microcytosis, but their diagnostic utility in population-based samples with ferritin-defined iron status is uncertain. We evaluated whether commonly used indices distinguish ferritin-defined iron-deficient from noniron-deficient microcytosis among women aged 18-49 years. METHODS:We analyzed the National Health and Nutrition Examination Survey (NHANES) 2015-2016, 2017-2018, and August 2021-August 2023 data among nonpregnant women aged 18-49 years with CBC, serum ferritin, and survey design variables. Among microcytic (MCV < 80 fL) women, iron-deficient microcytosis was defined as ferritin < 15 ng/mL and noniron-deficient microcytosis as ferritin ≥ 15 ng/mL. Mentzer, England-Fraser, Srivastava, and red cell distribution width (RDW) indices were evaluated at conventional cutoffs. Sensitivity analyses used ferritin < 30 ng/mL and C-reactive protein (CRP) restrictions of ≤ 5 and ≤ 3 mg/L. All estimates were survey-weighted. RESULTS:Among 3991 women, 507 had microcytosis. Ferritin-defined noniron-deficient microcytosis comprised 40.8% (95% CI, 35.5-46.2) and remained 37.0% and 33.6% after CRP restrictions of ≤ 5 and ≤ 3 mg/L. With ferritin ≥ 30 ng/mL, the corresponding proportions were 26.5%, 22.6%, and 20.2%. All four indices showed high sensitivity (92.9%-97.6%) but poor specificity (6.7%-35.3%). RDW had the highest specificity but still misclassified most noniron-deficient cases. CONCLUSIONS:Classic CBC indices had limited utility as standalone triage tools for ferritin-defined microcytosis. Ferritin-based assessment, interpreted in the relevant clinical and inflammatory context, should remain central. Persistent microcytosis without evidence of reduced iron stores should prompt diagnostic reconsideration and, when appropriate, hemoglobinopathy-aware evaluation.
INTRODUCTION:Thrombocytopenia has heterogeneous mechanisms, including peripheral destruction, consumption, and reduced marrow production. This study developed an unsupervised global complete blood count/research-use-only/cell population data (CBC/RUO/CPD) phenotyping model and evaluated post hoc whether thrombocytopenia severity and clinically adjudicated mechanisms were enriched within the resulting clusters. METHODS:Retrospective haematology analyser data from 254 289 unique specimens were analysed after cleaning 537 279 extracted rows. Exploratory missingness filtering retained 123 features and 173 851 complete-case specimens. The final model used a refined 41-feature CBC_EXTENDED complete-case dataset containing 241 591 specimens. Features were standardised, reduced using principal component analysis to 20 components explaining 92.94% of cumulative variance, and clustered using k-means. Thrombocytopenia thresholds and electronic health record (EHR) adjudicated primary mechanism labels were evaluated post hoc. RESULTS:The final global k-means model (k = 4) generated four broad haematological clusters comprising 14%-37% of specimens. Internal separation was modest but interpretable, with sampled silhouette 0.10, Davies-Bouldin 2.21, and Calinski-Harabasz 6536.92 in the fixed 60 000 specimen model comparison analysis. Cluster 3 showed progressive post hoc enrichment with thrombocytopenia severity, accounting for approximately 46%, 60%, and 71% of specimens with platelet counts < 150, < 100, and < 50 × 109/L, respectively. In 225 clinically annotated thrombocytopenic specimens, k-means k = 4 showed limited but non-random concordance with adjudicated mechanisms (purity 0.59; adjusted Rand index 0.14; normalised mutual information 0.18). CONCLUSION:Unsupervised global CBC/RUO/CPD clustering identified broad laboratory phenotypes with post hoc enrichment for thrombocytopenia severity and adjudicated mechanisms. These findings support underutilised analyser-derived parameters for laboratory phenotyping and hypothesis generation, not standalone diagnosis.
