INTRODUCTION:The myeloma patients with baseline normal/near-normal serum free light chain (SFLC) ratio have been reported to respond favorably to the treatment, unlike patients with extreme/abnormal SFLC ratio. We hypothesized that this normal SFLC ratio could serve as a biomarker for favorable cytogenetic and outcome profiles. MATERIALS AND METHODS:The flow cytometric immunophenotyping (FCM-IPT) and interphase FISH analysis of marrow aspirate were performed as per recommended guidelines, in all the newly diagnosed patients of plasma cell proliferative disorders, enrolled during this prospective study. Patients were risk-stratified as per ISS, R-ISS, and R2-ISS criteria. The outcome evaluation, in patients treated with standard 3-/4-drugs induction, was done at least 3 months after initiation of therapy. RESULTS:A total of 306 patients with plasma cell proliferative disorders were enrolled; myeloma patients were 240 (n_SFLC = 23/240, 9.6%) and (ab_SFLC = 217/240, 90.4%). IgG lambda M-protein was more significantly seen in (n_SFLC) patients. None of the patients in the (n_SFLC) group showed circulating blood plasma cells. The (n_SFLC) patients did not show the presence of TP53 gene deletion and relatively lacked other high-risk genetics. The outcome analysis showed that more patients in the (n_SFLC) group attained ≥ VGPR (88.9% vs. 65.4% of ab_SFLC). CONCLUSION:Based on this, we infer that the patients with a normal SFLC ratio are enriched with non-high-risk features and behave less aggressively.
INTRODUCTION:Improvements in treatment of patients with haemophilia A have meant that their quality of life has majorly improved. However, a disadvantage to these developments has come at a cost to the laboratory when monitoring patient Factor VIII (FVIII) levels and there is the potential for under- or overtreatment leading to clinical risk towards the patient. METHODS:A global study performed in 2023 to investigate the differences in FVIII assay results in a large cohort of laboratories has enabled users to understand the challenges that new products can cause. Five FVIII modified/extended half-life (EHL) products were studied by distributing spiked samples via 3 external quality assessment (EQA) schemes (ECAT, NEQAS and RCPAQAP). RESULTS:Participating laboratories used either one-stage assays (OSA), chromogenic assays (CA), or both methods when performing the assays. Most centres running the OSA used IL Hemosil Synthasil, Siemens Actin FS, Siemens Pathromtin SL, or Stago Cephalin/Kaolin/C.K. Prest reagents, and for the CA, the Chromogenix Coamatic FVIII kit and the Siemens chromogenic FVIII kit were utilised. CONCLUSION:The FVIII results submitted by participants showed that currently available OSA and CA do not provide consistent results in some products with both an under- and over-estimation of the expected recovery based on potency at either concentration level. Results for Afstyla Lonoctocog alfa suggest that centres were not clear on whether OSA results were before or after application of the correction factor (multiplication of initial result by 2).
INTRODUCTION:Elevated vitamin B12 concentration can be caused by supplementation, liver disease, kidney disease, or myeloid malignancies. Persistent, unexplained elevations of vitamin B12 can raise concern among patients and may lead to invasive diagnostic procedures, including bone marrow biopsy. A potential benign cause of this elevation is macro-B12, a complex of vitamin B12, transcobalamin, and immunoglobulins. Although not bioactive, this complex can cause elevated plasma vitamin B12 concentrations due to reduced clearance. METHODS:Polyethylene glycol (PEG) precipitation is a laboratory technique that can be used to support the suspicion of macro-B12. Here, a case is described in which a PEG precipitation could potentially have prevented an unnecessary bone marrow biopsy. In addition, the presence of macro-B12 was studied in a group of patients with and without myeloid malignancies. RESULTS:Macro-B12 was identified in 24% of 72 individuals with vitamin B12 > 1476 pmol/L. In one of these patients, a functional vitamin B12 deficiency was confirmed by an elevated methylmalonic acid (MMA) concentration. Macro-B12 was not detected in 8 patients with a myeloid malignancy. CONCLUSION:These findings suggest that, in patients with persistently elevated vitamin B12 concentrations and a low suspicion of a myeloid malignancy, PEG precipitation may help to explain the elevated vitamin B12 and prevent unnecessary diagnostic procedures including bone marrow punctures.
Vacuoles in hematopoietic cell precursors have garnered significant attention in recent years due to their association with a newly characterized clonal hematopoiesis with acquired mutations of ubiquitin like modifier activating enzyme 1 (ubiquitin-activating enzyme E1, UBA1 ) gene associated with clinical systemic autoinflammatory manifestations, known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. The cytomorphologic hallmark of this rare disorder is vacuolization involving the granulocytic and erythroid precursors. While affected vacuolated cells are seen in most patients, the mere presence of such is not entirely specific for VEXAS syndrome; therefore, an aggregate of clinicopathologic correlates is needed to help distinguish VEXAS syndrome from morphologic mimics and prompt appropriate confirmatory genetic testing. Vacuolated hematopoietic cells could be seen in varied reactive conditions and neoplastic and nonneoplastic hematologic disorders and affect different lineages and cell types. This article aims to review the spectrum of vacuolated hematopoietic cells and their disease-association including VEXAS syndrome, among others.
BACKGROUND:Accurate classification of novel globin gene variants is critical for the diagnosis and management of thalassaemia. The adaptation of ACMG/AMP guidelines for globin genes represents an important step toward standardising variant interpretation and enhancing clinical utility in the field. This study reports the haematological and molecular characteristics of a novel α2-globin variant identified in a Malay family. METHODS:A Malay family from Taiping, Perak, Malaysia, with a history of α-thalassaemia, was recruited for this study. The proband's phenotype was assessed through comprehensive haematological analysis and clinical evaluation. Known α-thalassaemia deletions and non-deletional mutations were screened using gap-PCR and ARMS-PCR. Sanger sequencing of the HBA genes was conducted to characterise the proband's genotype in detail. RESULTS:A novel pathogenic HBA2 variant was identified, expanding the known mutational spectrum of α-thalassaemia. This variant introduces a premature stop codon, occurs in trans with a known pathogenic allele associated with a significant clinical phenotype, segregates with the disease in the family, and is absent from major population databases. Based on haematological data, molecular findings, in silico predictions, and protein modeling, the variant meets the ACMG/AMP criteria for pathogenicity adapted for α-globin genes. We have designated this variant Hb Taiping, named after the location of its discovery. Its accurate classification is vital for carrier screening, genetic counselling, and prenatal diagnosis, thereby supporting improved clinical management. CONCLUSION:This study identifies and characterises a novel α-globin gene variant, Hb Taiping, in a Malay family with α-thalassaemia. The discovery contributes to the growing body of pathogenic mutations linked to α-thalassaemia and underscores the importance of precise variant classification for effective diagnosis, risk assessment, and genetic counselling.
Monocytes are key components of the Mononuclear Phagocyte System, crucial in immune defense, inflammation, and tissue repair. Accurate identification and classification of monocyte lineage cells are essential for diagnosing both reactive and clonal hematologic disorders. However, morphological criteria and nomenclature inconsistencies have hindered reproducibility, particularly with the rise of automated and AI-driven diagnostic tools. The International Council for Standardization in Haematology (ICSH) Monocyte Working Group (MWG) was convened to establish standardized morphological definitions, harmonize nomenclature for monocytes and their precursors, and evaluate their clinical utility as biomarkers. Data were collected from global laboratories, combined with an extensive literature review and consensus feedback from the ICSH General Assembly. The MWG proposes a three-category morphological classification: blasts and blast equivalents (monoblasts and promonocytes), immature monocytes, and mature monocytes. The recommendations reaffirm the importance of morphological analysis, cytochemical staining, and flow cytometry for accurate diagnosis. Emerging automated parameters, such as monocyte distribution width (MDW), and ratios like lymphocyte/monocyte (LMR) and neutrophil/monocyte (NMR), are also recognized as valuable adjunctive biomarkers. Cytogenetic and molecular results may also impact the utilization of monocytes as a biomarker. These harmonized recommendations aim to improve diagnostic accuracy, support the development of machine learning tools, and facilitate consistent reporting across laboratories worldwide.
INTRODUCTION:Accurate complete blood count (CBC) reference intervals are essential for neonatal care. However, existing reference intervals do not account for key clinical variables such as sex, postnatal age, gestational age at birth and corticosteroid exposure. This study aims to establish updated CBC reference intervals for neonates admitted to a Neonatal Intensive Care Unit (NICU) while evaluating the effects of these factors on the CBC. METHODS:In this retrospective cohort study, all neonates admitted to the NICU at the John Radcliffe Hospital (Oxford, United Kingdom) between January 2022 and January 2023 were eligible for inclusion. Routine CBCs were included if there was no suspicion of infection or necrotising enterocolitis, no recent surgical interventions and no signs of clinical deterioration. The effects of sex, gestational age at birth and postnatal age were assessed for 48 parameters of the CBC using multivariate ANOVAs. Reference intervals were calculated at the 95% level. RESULTS:Among 3490 CBC results from 587 neonates, 386 results from 196 neonates met inclusion criteria. Sex-related differences were observed in nine parameters. Gestational and postnatal age both significantly influenced 34 parameters. Reference intervals were produced for all 48 CBC parameters, with histograms and boxplots illustrating variations by sex, postnatal age and gestational age. Secondary analyses highlighted the effects of corticosteroid exposure. CONCLUSIONS:We present reference intervals for 48 neonatal CBC parameters, highlighting the influence of sex, postnatal age, gestational age at birth and corticosteroid exposure. These findings improve the interpretation of neonatal CBCs and propose criteria for defining a sufficiently healthy neonatal population for diagnostic research.
OBJECTIVES:Anemia is a significant global health concern, with hypochromic microcytic anemia being the most common type. Among its causes, β-thalassemia trait (β-TT) and iron deficiency anemia (IDA) share similar hematological features, making differentiation challenging. We aimed to develop machine learning (ML) models using routine red blood cell (RBC) indices to distinguish β-TT, IDA, and healthy cases. METHODS:A total of 8106 individuals (3378 healthy, 2696 IDA, 2032 β-TT) were included in this study. Six RBC parameters-RBC count, hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), and RBC distribution width (RDW-CV)-were used to train eXtreme gradient boosting (XGB), random forest (RF), and neural network (NN) models. The dataset was split into training (70%) and testing (30%) sets, with feature importance assessed via the Boruta algorithm. All statistical analyses were performed using R version 4.3.1 Statistical Language. RESULTS:All models achieved high accuracy (> 97%), with RF demonstrating superior performance (97.86% accuracy, 99.71% AUC). The most significant features contributing to the models are MCH for the XGB algorithm, MCV for the RF algorithm, and HGB for the NN algorithm. CONCLUSIONS:Our findings demonstrate that ML-based models could offer a promising tool for improving β-TT detection, optimizing clinical workflows, and enhancing resource utilization in hematology.
PURPOSE:Pediatric hypereosinophilia (HE) is rare, and its evaluation is challenging due to diverse etiologies and limited access to advanced laboratory testing in low- and middle-income countries (LMICs). METHODS:We retrospectively analyzed five children with HE (absolute eosinophil count > 5.0 × 109/L) evaluated at a tertiary center. Clinical, hematologic, and molecular findings were reviewed, and diagnostic timelines were compared between clonal and non-clonal cases. Based on these observations, a tiered, laboratory-driven diagnostic pathway adapted for LMICs was developed. RESULTS:Five patients (median age: 6 years; range: 1.5-10 years) were included. The median absolute eosinophil count (AEC) at presentation was 12.8 × 109/L (range: 6.5-21.5 × 109/L). Three cases (60%) were clonal eosinophilia-one PDGFRB-rearranged myeloid/lymphoid neoplasm and two acute myeloid leukemia subtypes [AML with CBFB::MYH11, AML with RUNX1::RUNX1T1]-and two cases (40%) were non-clonal [one Hyper-IgE syndrome with secondary hemophagocytic lymphohistiocytosis (HLH), one secondary eosinophilia (drug-induced, phenytoin/phenobarbitone)]. Clonal cases demonstrated higher leukocyte counts, earlier bone marrow and molecular testing, and shorter median time to diagnosis (6 vs. 14 days), enabling prompt initiation of imatinib or AML-directed therapy with remission in all. In contrast, non-clonal HE required sequential exclusion of clonal disease, delaying immunosuppressive or drug-withdrawal strategies. CONCLUSION:A structured, laboratory-driven algorithm beginning with blood counts, smear, and parasitic testing, and escalating to early bone marrow with cytogenetic/molecular studies for high-risk phenotypes, enabled timely identification of clonal HE while conserving resources in reactive cases. This LMIC-adapted pathway highlights laboratory turnaround time as a critical determinant of outcomes and provides a practical framework for pediatric HE evaluation.
OBJECTIVE:This study aims to analyze the expression patterns of CD371 in CD34+CD117+ bone marrow (BM) cells from patients with primary myelofibrosis (PMF) using flow cytometry (FCM), with a comparative evaluation against conventional antigens to assess its potential clinical utility for identifying aberrant immunophenotypes in PMF. METHODS:A retrospective analysis was conducted on BM samples from 26 PMF patients and 20 control individuals. We evaluated the proportions of CD34+CD117+ cells and basophils, as well as the expression profiles-including positive cell percentages, mean fluorescence intensity (MFI), and coefficient of variation (CV) of MFI-of CD371, CD34, CD117, CD13, CD33, CD123, CD38, HLA-DR, and CD7 within the CD34+CD117+ population. RESULTS:Control group exhibited consistent bimodal CD371 expression, while PMF samples showed three distinct patterns. PMF demonstrated significant differences vs. controls in CD371 MFI (p < 0.01), MFI CV (p < 0.01), and CD371+ cell proportion (p < 0.05). Within CD371+ cells, MFI and MFI CV also differed significantly (both p < 0.01). Significant differences (p < 0.01) were observed in: CD34+CD117+ proportion, CD34 MFI/MFI CV, basophil proportion, CD38dim+ proportion/CD38 MFI/MFI CV. No significant differences were observed for CD13+ cell proportion (p = 0.154) or CD13 MFI (p = 0.835), though the MFI CV was significantly different (p < 0.01). CD33+ cell proportion (p < 0.05) and CD33 MFI (p < 0.05) showed significant differences, while the MFI CV did not (p = 0.276). CONCLUSION:This study provides the first characterization of CD371 expression patterns in PMF, showing significantly different expression profiles compared to controls. While CD371 demonstrated comparable performance to standard markers (CD34/CD38/CD13/CD33) and showed better discrimination than CD123/HLA-DR, these preliminary findings suggest its potential utility for: (1) identifying abnormal CD34+CD117+ populations in PMF, and (2) possible integration into routine clinical workflows, pending further validation in larger cohorts.
OBJECTIVES:Early diagnosis of suspected sepsis is crucial to improve patient survival. Cell population (CP) data, a set of leucocyte research parameters from hematology instruments, has a potential as markers for infection. The aim of this study was to investigate the diagnostic accuracy for infection of CP variables from Sysmex XN instruments in patients with suspected sepsis in the emergency department (ED). METHODS:Adult patients with suspected sepsis in the ED were included. CP variables, C-reactive protein (CRP), and post hoc assessments of infection were recorded. Logistic regression and machine learning methods were used to develop multivariable models, which were evaluated by area under the receiver operating curve (AUC) and calibration plots. RESULTS:The development cohort and the validation cohort consisted of 600 and 656 patients, respectively. Univariate analyses revealed that complexity in monocytes (MO-X); AUC of 0.78 (0.74, 0.82), reactivity intensity of neutrophils (NEUT-RI); 0.72 (0.67, 0.76), and CRP 0.87 (0.84, 0.90) had the highest diagnostic accuracy for infection. A final multivariable model (the optimal model) using Multilayer perceptron (MLP), including MO-X, NEUT-RI, monocyte size (MO-Z), and neutrophil size (NE-FSC), had an AUC of 0.86 (0.85, 0.87) in the development cohort and 0.78 (0.74, 0.82) in the validation cohort with reasonable calibration. Including CRP in this model further improved accuracy and calibration. CONCLUSIONS:Sysmex CP variables may help diagnose infections in the ED. However, the lack of well-described calibration procedures and quality assurance for non-IVD approved CP variables is an impediment to clinical implementation.
INTRODUCTION:Multiple myeloma (MM) is an incurable clonal B-cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow (BM). Many pieces of evidence indicate that hypoxia promotes MM progression, but the underlying mechanisms are not well known. METHODS:We analyzed gene expression profiles of 3 MM cell lines under hypoxia and the MMRF CoMMpass project. We validated the expression patterns of hypoxia-associated genes (HAGs) in CD138+ BM cells from MM patients at different stages. Single-cell RNA sequencing data were used to analyze the performance of HAGs in the BM microenvironment. RESULTS:We identified 17 HAGs differentially expressed in three MM cell lines under hypoxia. While in the MMRF project, we identified 92 differentially expressed HAGs in newly diagnosed MM patients. MM cell lines and the MMRF project shared 9 HAGs, including ADM, BNIP3L, EGLN1, FAM162A, HMOX1, PDK1, PLOD1, STAT5B, and TFRC. Notably, 8 of them were significantly associated with the overall survival of MM patients, and 6 were significantly associated with the MM patient survival in the first year after diagnosis. Then, hypoxia pressure scores calculated using these genes displayed significant differences between MM patients and healthy individuals. Further, we validated the expression patterns of HAGs using another cohort data and performed qRT-PCR using our own samples, and the results confirmed severe hypoxia existed in plasma cells and other cell types of the BM microenvironment of MM patients compared to healthy individuals. CONCLUSION:Taken together, our findings may contribute to the treatment and prognosis prediction of MM patients.
INTRODUCTION:Atypical lymphocytes (ALYs) are activated lymphocytes with distinct morphological characteristics, often observed in various infections, autoimmune diseases, drug reactions, and malignancies. Their appearance may resemble leukemic or lymphoma cells, making it essential to differentiate ALYs, particularly in patients with hematological malignancies. With the advent of T-cell engagers (TCEs), a novel class of immuno-oncology drugs, this study aimed to investigate their effect on peripheral blood profiles, including ALYs. METHODS:We retrospectively analyzed complete blood count (CBC) data and peripheral blood morphology from 28 patients enrolled in clinical trials of various TCEs targeting multiple myeloma and B-cell lymphomas. The drugs studied included cevostamab, linvoseltamab, glofitamab, teclistamab, talquetamab, elranatamab, and epcoritamab. RESULTS:A transient increase in ALYs was observed in 11 of the 28 patients treated with TCEs. This was confirmed by changes in cell morphology and flow cytometric parameters obtained from the CBC analyzer. ALY elevation appeared to be influenced by drug type, administration route, and combination therapies. In addition, a sudden and transient decrease in both monocytes and lymphocytes was noted in peripheral blood following cevostamab treatment. CONCLUSION:The observed increase in ALYs likely reflects immune activation induced by TCEs. Understanding ALY dynamics during TCE treatment is crucial for clinicians and pathologists when interpreting patient test results. Furthermore, ALY testing may serve as a potential marker for predicting the effectiveness of TCE therapies.
