Flow cytometric analysis of leukocytes and red blood cells (RBCs) for loss of GPI-linked proteins is the gold standard for the diagnosis of PNH. Assessment of mature RBCs is often confounded by recent transfusions and the variable survival of normal versus PNH erythrocytes. Analysis of immature RBCs (iRBCs) provides a more accurate reflection of the underlying marrow clone size compared with mature RBCs (mRBCs). CD71 (transferrin receptor) serves as a robust marker for identifying iRBCs, thereby enabling the evaluation of PNH clones. This approach is still under investigation and has not yet been incorporated into the guidelines. We evaluated it in a large cohort of patients to validate its utility. FCM-based screening of leukocytes was carried out using a single-tube five-color panel (CD45-V500, FLAER-AF488, CD157-PE, CD15-PC 5.5, CD64-APC). The three-color RBC tube (CD235a-FITC, CD59-PE, CD71-APCH7) was used only in cases in which the leukocyte PNH clone size was >1%. Clone sizes across different cell populations, CD59 staining intensity, and the ability to distinguish among normal RBCs and type II and type III clones were compared between mRBCs (CD71-) and iRBCs (CD71+). A total of 782 samples were screened over a period of 24 months for various indications, including AA, non-immune hemolytic anemia, and unexplained thrombosis. PNH clones with a leukocyte clone size >1% were detected in 120 samples. The iRBC-PNH clone size was significantly higher than the mRBC-PNH clone size (median [range]: 49.9% [0.8-97.3] vs. 12.0% [0-86.0]; p = 0.002). The iRBC-PNH clone size corresponded closely with those of neutrophils and monocytes (median [range]: 49.9% [1-97.3] vs. 54.5% [1-99.5] vs. 58.7% [6.3-98.5], respectively). The MFI of CD59 in iRBCs was higher than that in mRBCs (8996 vs. 5687; p < 0.001). Normal RBCs and type II and III clones were better delineated in iRBCs than in mRBCs. Among cases with clinical evidence of hemolysis and/or thrombosis, the iRBC-PNH clone size reflected the disease burden better than the mRBC-PNH clone size (median [range]: 80.4% [20-95.6] vs. 26.6% [1.1-85.6]). This study validates the role of CD71-based analysis of iRBC-PNH clones, which was found to improve the detection, delineation, and sizing of PNH RBC clones and to correlate better with leukocyte clone sizes and clinical features. Therefore, incorporation of CD71 into high-sensitivity FCM-based PNH screening is suggested.

Sensorineural hearing loss (SNHL) is an underrecognized manifestation in chronic myeloid leukemia (CML), particularly in the setting of hyperleukocytosis. Its prevalence, pathophysiology, and response to tyrosine kinase inhibitor (TKI) therapy remain poorly characterized. To assess the prevalence, severity, and reversibility of SNHL in treatment-naïve CML patients and evaluate associations with leukocyte count and disease risk. In this prospective study, 102 newly diagnosed CML patients with total leucocyte count (TLC) > 50 × 109/L underwent audiological evaluation at baseline, 3 months, and 6 months post-initiation of imatinib. Age- and sex-matched healthy controls were included for comparison. SNHL was identified in 45.1

Minimal residual disease (MRD) assessment using multiparametric flow cytometry (MFC) in acute myeloid leukaemia (AML) is a well-established predictor of relapse and survival outcome. This study aimed to evaluate the prognostic significance of MFC-based MRD assessment in AML patients managed in a real-world tertiary care setting. In this retrospective observational study, the impact of MFC-based MRD on overall survival (OS) was analysed. MRD was assessed at four predefined time points: post-induction (PI), post-consolidation (PC), pre-transplant (PT), and day 100 post-transplant (AT). Detection of MRD employed both leukaemia-associated immunophenotype (LAIP) and different-from-normal (dfN) approaches using a two-tube, 10-colour panel. Aberrant antigen expression patterns and their relevance across ELN 2022 risk categories were also examined. A total of 206 MRD assessments were performed in 125 patients, with 45 MRD-positive samples identified in 30 patients (33.8

BACKGROUND:Chronic myeloid leukaemia (CML) is an infrequent myeloproliferative neoplasm in the paediatric population as compared to adults. Despite vast progress in understanding the disease biology and therapy of CML-chronic phase (CML CP), the applicability of risk scoring systems in practice and prognostic factors in children remain grey areas. Hence, we tried to analyse disease characteristics, molecular response to frontline imatinib therapy and its clinical predictors along with long-term outcomes in our population. MATERIALS AND METHODS:In this study, we retrospectively analysed 104 paediatric patients aged ≤ 18 years, diagnosed with CML CP, treated at our centre between 2007 and 2024. Their baseline demographic profile, clinical characteristics, haematological parameters and molecular transcripts as diagnosed by RT-PCR were recorded. The follow-up response assessments, including haematological response, molecular response and long-term survival, were collected from medical records. Risk scores were computed and correlated with clinical outcomes. RESULTS:Our study included 104 paediatric patients presenting with CML CP. Splenomegaly was a universal feature with a median size of 10 cm below costal margin. The majority of patients belonged to the low-risk group according to Sokal, EUTOS and ELTS systems. Imatinib was initiated in all of them and was followed up for a median duration of 72 months. Overall, 95.2% patients achieved complete hematologic response (CHR) at 3 months. A total of 42.5% (43/101) patients achieved MMR at 12 months and 54.4% (55/101) patients achieved MMR at 18 months. Sokal scoring system and EMR at 3 months were significantly correlated with the achievement of MMR at 12 months. A total of 11 patients progressed to blast crisis and 10 patients expired in due course of the disease. The 2- and 10-year PFS was 96% and 82%, respectively. CHR at 3 months was significantly predictive of PFS. None of the scoring systems had predictive value for PFS. CONCLUSION:This is one of the largest reported data on Indian paediatric CML CP patients with long-term outcomes. The dynamics of adult CML, including risk scores, do not perfectly fit the scenario of paediatric patients. Hence, further studies and newer strategies are required for optimal management of paediatric CML patients.

Bone marrow sampling [including bone marrow aspirate (BMA), trephine biopsy touch imprint (BMI), and the bone marrow trephine biopsy (BMBx)] is a very important test in the diagnosis and/or follow-up monitoring of both hematological and many non-hematological conditions. A synoptic report for BMA/BMI cytology aims to bring homogeneity in reporting among pathologists/laboratory hematologists/hematopathologists across academic and practicing centres, and ensure completeness in communicating with clinicians with actionable suggestions. The essence of such reporting format is to bring out the accuracy in reporting that will incorporate treating physician/hematologist’s perspective, essential patient related clinical information gathered and documented during bone marrow sampling, careful interpretation of freshly prepared and stained peripheral blood smear (PBS) morphology, recent complete blood count parameters, and meticulous evaluation of hematopoietic and non-hematopoietic elements in the bone marrow. The integrated PBS-BMA-BMI report should be made available in a time bound manner maintaining reasonable turnaround time. It should be clear and comprehensive, and every attempt should be made to offer preliminary vital diagnostic information regarding the underlying disease process wherever possible; suggest requisite routine and/or specialized ancillary testing; and finally guide the treating physician for further plan of patient management. The BMA-BMI reporting should be followed-up with that of BMBx, preferably, by the same reporting personnel. This ICH-ISHBT taskforce consensus document on BMA and BMI synoptic reporting intends to address the above issues, bring out homogeneity, and improve the quality of patient care at par with national and international standards.

BACKGROUND:The classical or modified Nijmegen-Bethesda assay measures FVIII inhibitor titers and requires testing residual FVIII levels at multiple serial dilutions, making it prohibitively expensive in resource-constrained settings. The study aimed to develop a cost-effective strategy to predict inhibitor titers (Bethesda units [BU]) in patients with hemophilia A (HA) using the inhibitor screening assay. METHODS:Inhibitor screening and Bethesda assay were performed on samples of suspected HA with inhibitors. The laboratory data were analyzed to develop a model to predict inhibitor titers from inhibitor screening (normalized ratio of APTT values of the fresh mix [FM] or incubated mix [IM]). The model was then prospectively validated on new inhibitor-positive samples. RESULTS:One hundred and four samples from 83 patients with HA and inhibitors were analyzed. A linear regression analysis was performed, and the normalized APTT ratio of FM (R2 = 0.815) proved to be highly predictive of inhibitor titers (BU) compared with IM (R2 = 0.536). A predictive model was developed based on the normalized APTT ratio of FM values. This predictive model was then validated on a new cohort of 27 inhibitor-positive samples, where the predicted inhibitor titers strongly correlated with the observed titers (R2 = 0.843). CONCLUSION:The FM of the screening assay serves as a potential indicator of the expected inhibitor titers (BU) and guides the selection of dilutions likely to yield near-50% residual FVIII activity for estimating the Bethesda titers. This approach substantially reduces assay costs, particularly in cases with > 2 BU. Additional prospective studies are needed to validate these results.

Bone marrow aspirate and trephine biopsy examinations are indispensable components of the diagnostic workup for a wide spectrum of hematological and non-hematological disorders. An ideal bone marrow report should provide a comprehensive and integrated evaluation of all available specimens, including aspirate smears, imprint (touch) preparations, cytochemistry, and trephine biopsy sections. It should also indicate the need for further appropriate ancillary investigations such as immunophenotypic, cytogenetic, and molecular studies when indicated. These findings should be interpreted in conjunction with the clinical history and other relevant laboratory data to ensure an accurate, meaningful, and clinically relevant diagnosis, facilitate therapeutic decision-making, and support optimal patient management. This document provides practical recommendations for developing a standardized synoptic reporting format for bone marrow biopsy. The proposed framework is intended to be practical, reproducible, and readily implementable, thereby promoting uniformity, consistency, and quality in bone marrow reporting across India.

BACKGROUND:Aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome marked by pancytopenia and a hypocellular, fatty bone marrow. Growing evidence indicates that intrinsic abnormalities in bone marrow-derived mesenchymal stem cells (BM-MSCs), including inadequate hematopoietic support, cellular senescence, chronic inflammation, and enhanced adipogenic differentiation, contribute to disease pathophysiology. However, the epigenetic mechanisms underlying these abnormalities remain poorly understood. This work aims to define the epigenetic landscape of AA BM-MSCs and investigate key epigenetic regulators associated with niche failure. METHODS AND RESULTS:RNA sequencing data from AA patients and healthy controls were analyzed using bioinformatics methods, including differential gene expression analysis, Gene Ontology (GO) analysis, and Reactome pathway enrichment analysis. Principal component analysis revealed a clear separation between AA and control samples. Differential expression analysis identified 713 disrupted epigenetic regulators in AA BM-MSCs. Functional enrichment analysis indicated significant changes in pathways including chromatin remodelling, stem cell maintenance, DNA damage response, cellular senescence, inflammation, and lineage commitment. Quantitative real-time PCR validation confirmed overexpression of SETD1A, MLL1, EZH2, KDM3A, and PRMT1, and downregulation of DNMT3A, KDM2B, EHMT1, and KDM5C, consistent with the transcriptomic results. CONCLUSIONS:Our data show that AA BM-MSCs exhibit broad epigenetic dysregulation, which may contribute to bone marrow niche failure, chronic inflammation, senescence-associated changes, and reduced hematopoietic support. These findings provide novel insights into the epigenetic basis of AA pathobiology and suggest potential treatment targets to restore the function of the bone marrow microenvironment.

BACKGROUND:Dasatinib, a potent second-generation tyrosine kinase inhibitor (TKI), is highly effective in chronic myeloid leukemia in chronic phase (CML-CP) resistant to imatinib at standard dosing (100 mg daily), but is often limited by adverse events. Emerging evidence suggests low-dose dasatinib (50 mg daily) may maintain efficacy with improved safety, but data in imatinib-resistant CML-CP remain limited. AIMS:To evaluate the efficacy and safety of low-dose dasatinib (50 mg daily) in patients with imatinib-resistant CML-CP and to identify predictors of treatment response and disease progression. METHODS AND RESULTS:This retrospective cohort study included 53 adults with imatinib-resistant CML-CP treated with low-dose dasatinib at a tertiary center in Northern India (2002-2025). Early molecular response (EMR), major molecular response (MMR), deep molecular response (DMR), progression-free survival (PFS), overall survival (OS), and adverse events were assessed. Multivariate Cox regression identified predictors of poor response and disease progression. Among 53 patients (median age 50 years), 41.5% achieved MR4.5, 20.8% MR4.0, and 15.1% MMR without DMR. Prior loss of MMR on imatinib significantly correlated with a superior response to dasatinib (p = 0.002). TKD mutations were present in 32.1%; the T315I mutation, high ELTS risk, and baseline BCR-ABL1 ⟩ 100% independently predicted poor response. Clinically significant adverse events occurred in 49.1%, primarily cytopenias and pleural effusion. Among our cohort, 22.6% required a TKI switch due to inadequate response and 7.5% due to intolerance. CONCLUSION:Low-dose dasatinib is effective and tolerable in imatinib-resistant CML-CP, with nearly two-thirds achieving DMRs. Predictive biomarkers (T315I mutation, high ELTS risk, high baseline BCR-ABL1) can guide dose optimization.

Examination of the peripheral blood smear (PBS) is an integral component of hematologic evaluation that complements the complete blood count (CBC) and red blood cell indices in the initial assessment of most patients with suspected or established hematologic disorders. A well-prepared PBS provides critical diagnostic information regarding the formed elements of blood, often suggesting specific disease categories, guiding further investigations, and in selected conditions, prompting urgent clinical intervention. The PBS also serves additional roles: it is an important quality assurance tool for validating automated analyzer results, helps to identify spurious counts and instrument artifacts, and supports competency-based training in morphology for resident doctors and fellows. Manual microscopic review of Romanowsky-stained smears remains the reference standard for detailed morphological assessment of a blood film, particularly in complex or flagged cases. This remains true despite major advances in automated hematology analyzers and more recently, digital morphology platforms. Given the central role of the PBS and the variability that currently exists in PBS reporting formats and terminology, there is a need for harmonized, practical guidance on synoptic reporting that can be applied across diverse laboratory settings. Synoptic reporting, i.e., the use of structured headings and checklists to capture essential morphological and interpretive elements, and not missing important findings has been shown to improve report completeness, consistency, educational value, and turnaround time, while reducing typographical and omission errors. These recommendations aim to address this gap by proposing a structured, yet flexible, synoptic format for PBS reporting that aligns with the current state of knowledge and with international recommendations for morphological nomenclature and grading.

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: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.

Treatment of paroxysmal nocturnal hemoglobinuria (PNH) patients with a large-size clone is a major challenge in low and middle-income countries. In the absence of complement inhibitors, the response rates with available therapy have not been evaluated in the Indian subcontinent. Retrospective data for patients with a large PNH clone >12 years of age were collected from 2004 to 2024 and were further classified as: Group 1: Classical PNH (c-PNH); Group 2: PNH with aplastic anemia (AA) occurring simultaneously: PNH(L)/AA-s; Group 3: AA preceding the evolution of a Large PNH clone: PNH(L)/AAp. A total of 211 patients were identified with a mean age of 33 years. Of these patients, survivorship status was known for 163 (77.2

Alloreactive KIR-B haplotypes mediate potent anti-tumor and anti-microbial effects and can improve the prognosis of haploidentical hematopoietic cell transplantation (HHCT) but there is paucity of this data in the Indian population. We aimed to evaluate the prevalence of KIR-B haplotypes and their alloreactivity in HHCT donors at our center. A total of 119 individuals (haploidentical donors, (n = 93); and patients, n = 26) from 26 families were included in this study. The KIR genotyping of the donors and HLA-B, as well as HLA-C genotyping of donor-recipient pairs, was done by polymerase chain reaction with sequence-specific primer genotyping assay. The KIR B-content score of donors was calculated using the online donor KIR B-content group calculator of the Immuno Polymorphism Database (IPD)( www.ebi.ac.uk/ipd/kir/donor_b_content ). The alloreactivity of KIR was determined using an online KIR ligand matching calculator of IPD ( www.ebi.ac.uk/ipd/kir/matching/ligand ). Haploidentical donors were siblings and parents in 64

Autologous stem cell transplantation (ASCT) remains a cornerstone in the management of plasma cell dyscrasias, particularly in younger patients, despite advancements in novel therapeutic agents. However, real-world outcomes in resource-limited settings like India remain underreported. We conducted a single-center, retrospective analysis of 67 patients with plasma cell dyscrasias who underwent ASCT between December 2012 and October 2024 at a tertiary care center in Northern India. Clinical characteristics, induction regimens, transplantation details, and outcomes were evaluated. Relapse-free survival (RFS) and overall survival (OS) were calculated using Kaplan-Meier analysis, and risk factors for relapse were identified through logistic regression. The median patient age was 50 years, with a male predominance (73.1%). Multiple myeloma accounted for 88.1% of cases. Induction regimens varied, with CyBorDex being most common. Median time to neutrophil and platelet engraftment was 10 and 12 days, respectively. Febrile neutropenia occurred in 68.9% of patients, and 30-day transplant-related mortality was 7.5%. At a median follow-up of 4 years, the 5-year OS and RFS were 74.5% and 53%, respectively. High-risk cytogenetics and positive day 100 PET-CT showed trend towards higher risk of relapse. ASCT remains effective in improving survival and deepening response in plasma cell dyscrasias, even in resource-constrained settings. Relapse rates and transplant-related mortality are higher than Western cohorts, underscoring the need for improved supportive care and broader access to maintenance therapies in India.