Peripheral blood quantitation of CD26 positive leukemic stem cells as a predictor of tyrosine kinase inhibitor response in chronic myeloid leukemia.

INTRODUCTION: Leukemic stem cells (LSCs) are the transcriptionally low/silent cells which are resistant to the tyrosine kinase inhibitor. These have been found to play a pivotal role in disease relapse in chronic myeloid leukemia (CML) cases. The present study evaluated the correlation of absolute CML-LSC count in the peripheral blood (PB) at diagnosis and achievement of major molecular response (MMR) at 12 months in patients of CML-CP. METHODS: This was a prospective, observational, non-interventional single center study including newly diagnosed adult (>18 yrs) CML-CP patients. Absolute CD26 + CML-LSC quantification was done by multiparametric flow cytometry. Patients were treated with Imatinib treatment and subsequently monitored at 3-month intervals for BCR::ABL transcript levels. MMR was defined as a BCR::ABL1 transcript level of less than 0.1% on international scale. RESULTS: A total of 89 patients were enrolled in the study out of which 40.5% achieved MMR at 12 months. There was a significant difference in the median absolute CML-LSC count of the patients who achieved MMR at 12 months as compared to those who did not (58.5 vs 368.1 cells/µL; p value <0.001). Using a ROC analysis, a count of <165.69 CML LSC/µL was identified to have a sensitivity of 83.8% and specificity of 72.4%, in predicting the MMR at 12 months. CONCLUSION: Absolute CML-LSC count at diagnosis in the PB predicts the MMR achievement at 12 months. An absolute count of less than 165 cells/µL is highly predictive of achieving MMR at 12 months.

Role of miRNAs in T-cell activation and Th17/Treg-cell imbalance in acquired aplastic anemia.

BACKGROUND: Altered T-cell repertoire with an aberrant T-cell activation and imbalance of the Th17/Treg cells has been reported in acquired aplastic anemia (aAA). miRNAs are well known to orchestrate T-cell activation and differentiation, however, their role in aAA is poorly characterized. The study aimed at identifying the profile of miRNAs likely to be involved in T-cell activation and the Th17/Treg-cell imbalance in aAA, to explore newer therapeutic targets. METHODS: Five milliliters peripheral blood samples from 30 patients of aAA and 15 healthy controls were subjected to flow cytometry for evaluating Th17- and Treg-cell subsets. The differential expression of 7 selected miRNAs viz; hsa-miR-126-3p, miR-146b-5p, miR-155-5p, miR-16, miR-17, miR-326, and miR-181c was evaluated in the PB-MNCs. Expression analysis of the miRNAs was performed using qRT-PCR and fold change was calculated by 2-ΔΔCt method. The alterations in the target genes of deregulated miRNAs were assessed by qRT-PCR. The targets studied included various transcription factors, cytokines, and downstream proteins. RESULTS: The absolute CD3+ lymphocytes were significantly elevated in the PB of aAA patients when compared with healthy controls (p < 0.0035), however, the CD4:CD8 ratio was unperturbed. Th17: Treg-cell ratio was altered in aAA patients (9.1 vs. 3.7%, p value <0.05), which correlated positively with disease severity and the PNH positive aAA. Across all severities of aAA, altered expression of the 07 miRNAs was noted in comparison to controls; upregulation of miR-155 (FC-2.174, p-value-0.0001), miR-146 (FC-2.006, p-value-0.0001), and miR-17 (FC-3.1, p-value-0.0001), and downregulation of miR-126 (FC-0.329, p-value-0.0001), miR-181c (FC-0.317, p-value-0.0001), miR-16 (FC-0.348, p-value-0.0001), and miR-326 (FC-0.334, p-value-0.0001). Target study for these miRNAs revealed an increased expression of transcription factors responsible for Th1 and Th17 differentiation (T-bet, RORϒt, IL-17, IL-6, and IFN-ϒ), T-cell activation (NFκB, MYC, and PIK3R2), downregulation of FOX-P3, and other regulatory downstream molecules like SHIP-1, ETS-1, IRAK-1, TRAF-6, and PTEN. CONCLUSION: The study for the first time highlights the plausible role of different miRNAs in deregulating the Th17/Treg-cell imbalance in aAA, and comprehensively suggest the role of altered NF-kB and mTOR pathways in aAA. The axis may be actively explored for development of newer therapeutic targets in aAA.

Ligand-mediated electron transport channels enhance photocatalytic activity of plasmonic nanoparticles.

Photoexcitation of noble metal nanoparticles creates surface plasmons which further decay to form energetic charge carriers. These charge carriers can initiate and/or accelerate various chemical processes at nanoparticle surfaces, although the efficiency of such processes remains low as a large fraction of these carriers recombine before they can reach the reaction sites. Thus efficient utilization of these charge carriers requires designing nanostructures that promote the separation of charges and their transport toward the reaction sites. Here we demonstrate that covalently bound surface-coating ligands with suitable orbital alignment can provide electron transport channels boosting hot electron extraction from a gold nanostructure leading to a huge enhancement in the rate of hydrogen evolution reaction (HER) under NIR excitation. A (p)Br-Ph-SH substituted gold nanoprism (AuTP) substrate produced ∼4500 fold more hydrogen compared to a pristine AuTP substrate under 808 nm excitation. Further experimental and theoretical studies on a series of substituted benzene-thiol bound AuTP substrates showed that the extent of the ligand-mediated HER enhancement depends not only on the polarity of the ligand but on the interfacial orbitals interactions.

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis.

Erythropoiesis is a highly regulated process and undergoes several genotypic and phenotypic changes during differentiation. The phenotypic changes can be evaluated using a combination of cell surface markers expressed at different cellular stages of erythropoiesis using FACS. However, limited studies are available on the in-depth phenotypic characterization of progenitors from human adult hematopoietic stem and progenitor cells (HSPCs) to red blood cells. Therefore, using a set of designed marker panels, in the current study we have kinetically characterized the hematopoietic, erythroid progenitors, and terminally differentiated erythroblasts ex vivo. Furthermore, the progenitor stages were explored for expression of CD117, CD31, CD41a, CD133, and CD45, along with known key markers CD36, CD71, CD105, and GPA. Additionally, we used these marker panels to study the stage-specific phenotypic changes regulated by the epigenetic regulator; Nuclear receptor binding SET Domain protein 1 (NSD1) during erythropoiesis and to study ineffective erythropoiesis in myelodysplastic syndrome (MDS) and pure red cell aplasia (PRCA) patients. Our immunophenotyping strategy can be used to sort and study erythroid-primed hematopoietic and erythroid precursors at specified time points and to study diseases resulting from erythroid dyspoiesis. Overall, the current study explores the in-depth kinetics of phenotypic changes occurring during human erythropoiesis and applies this strategy to study normal and defective erythropoiesis.

Role of T Lymphocyte Activation Profile in Predicting SARS-CoV-2 Severity: Experience from Tertiary Care Centre of North India.

Background: Immune dysregulation plays a key role in determining COVID-19 disease severity. We aimed to analyze the T cell activation profile in COVID - 19 cases and its predictive role in disease severity and outcome. Material & methods: This was a prospective observational pilot study from a tertiary care COVID-19 hospital. Peripheral blood samples obtained between the fifth and seventh day of COVID-19 illness, were subjected to lymphocyte subset analysis using multicolor flowcytometry using a single tube, 8 antibodies (CD45, CD19, CD3, CD4, CD8, CD38, HLADR, and CD56) analysis. Correlation between lymphocyte subset analysis and clinical profile was determined. Results: 26 patients including 11 with mild disease and 15 with severe disease were enrolled. The median age was 58 years (range: 33-81), with a male: female ratio of 1.36:1. Significant lymphopenia was observed in the severe group compared to the mild group (p < 0.02). The absolute numbers of CD3+, CD4+, CD8 + T cells, B cells, and NK cells were significantly reduced in the severe group as compared to the mild group (p < 0.05). In patients with severe disease, the proportion of CD8 + and CD4 + T cells were significantly higher than those in patients with mild disease (p = 0.0372). Using ROC analysis, a CD4:8 T cell ratio of ≥ 2.63 and an activated (CD38 + HLA-DR+) CD8 T cell proportion of > 15.85% of the total CD8 T cell population, significantly determined the severe disease category. Conclusions: Severe COVID-19 is associated with severe lymphopenia, altered CD4/CD8 ratio and markedly increased CD8 T cell activation profile. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01558-6.

An Analysis of M-protein in Plasma cell Dyscrasia Patients Identifies that IgG Lambda Subtype is More Commonly Associated with Normal Serum Free Light Chain (SFLC) Ratio.

The determination of monoclonal protein (M-protein) by SPE, IFE and SFLC assay is fundamental in the diagnosis of Plasma cell proliferative disorder (PCPD). In the present study, we seek to assess the diagnostic performance and concordance of these three techniques in un-treated PCPD patients. All new patients with dysproteinemia and/or suspected PCPD were included in this retrospective observational study. The baseline parameters were retrieved from electronic medical records. SPE was performed on gel electrophoresis system; monoclonal component was identified by IFE. SFLC assays were performed by nephelometry using a latex-enhanced immunoassay. Total 402 patients of PCPD were included (10.9% of MGUS/SMM and 89.1% of multiple myeloma). The combination of SPE + rSFLC (ratio of kappa/lambda light chain) and SPE + IFE + rSFLC was able to detect M-protein across all subgroups of patients. In 61 patients, rSFLC values were within normal range (54.5% of MGUS/SMM and 10.3% of MM) and was more commonly seen with IgG lambda M-protein (57.4% vs. all-others). The median dFLC value, among these patients, was higher for MM than MGUS/SMM patients (23.8 vs. 14.4 mg/L, respectively). The combination of SPE and rSFLC can be reliably used to detect M-protein in PCPD patients. In a small subgroup of MM patients, despite the presence of an intact immunoglobulin (M-protein), the rSFLC is not abnormal. Historically, these patients should respond better to treatment. However, a further follow-up analysis with more number of such patients would be advantageous for better understanding.

CD26 expression on circulating CD34+/CD38- progenitor population is a specific and reliable tool for the rapid flow cytometric diagnosis of chronic myeloid leukemia-A single-center validation study.

BACKGROUND: Recently, CD26 have been identified as one of the promising and specific marker for the identification of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML). METHODS: This was a prospective, observational validation study. Peripheral blood (PB) samples from suspected cases of CML and other hematolymphoid neoplasm were evaluated for the expression of CD26 on stem cells (SC) (CD45 dim/CD34+/CD38-) fraction by flow cytometry (FCM) using a single tube four-color antibodies cocktail: CD45-V500 /CD26-PE/CD34-PerCPcy5.5/CD38-APC-H7. The diagnosis of CML was confirmed using cytogenetics and/or molecular studies. Additionally, 12 paired PB and bone marrow (BM) samples of CML cases were compared for the proportion of CD26+ LSCs. RESULTS: Expression of CD26 on the SC fraction was invariably noted in all cases (116/116) of CML, irrespective of the disease phase and transcript type. None of other neoplasm (0/26), including the Ph + ALLs expressed CD26. Proportion of SCs expressing CD26 was variable with a median (range) proportion being 61.3% (7.6%-98.6%). Evaluation of paired PB and BM samples showed similar proportion of CD26 + LSCs (R2 : 0.969). CONCLUSION: We confirmed that FCM evaluation of CD26 expression in the PB LSCs is a rapid and specific tool for CML diagnosis. Its utility as a marker for residual disease evaluation can also be explored in the future.

Leonine facies: A unique presentation of T-prolymphocytic leukemia.

We report a 52-year-old man who presented with erythroderma and nodular lesions on face manifesting as "Leonine facies". He had impaired sensation over the face and was initially diagnosed to have lepromatous leprosy and was treated with antileprosy drugs. Investigations showed a total Leukocyte count of 550 X 109/l with 90% atypical lymphoid cells with prominent central nucleolus suggestive of prolymphocytes. On flow cytometry, these cells were positive for cytoplasmic CD3, CD2, CD5, CD7, CD4, and CD38 (dim) and were negative for CD1a and TdT and diagnosis of T-prolymphocytic leukemia was made.

Waldenström Macroglobulinemia: Clinico-pathological Profile and Treatment Outcomes of Patients from a Tertiary Care Centre of North India.

Waldenstorms Macroglobulinemia (WM) is a rare mature B cell neoplasm characterized by a lymphoplasmacytic lymphoma and an IgM monoclonal protein. It is managed by Rituximab based chemotherapy. A single-centre retrospective study was carried out to analyse the clinical presentation, laboratory features, and treatment outcomes of all consecutive patients of WM, diagnosed over a period of 86 months. First-line treatment regimens included RCD (Rituximab/Cyclophosphamide/Dexamethasone), BDR (Bortezomib /Dexamethasone/ Rituximab) and (Lenalidomide/Dexamethasone). A total of 26 patients of WM were diagnosed during this period, with a median age of 65 years. Majority (89%) of these patients were of intermediate (47%) to high risk (42%). An overall response rate of 76.4% was achieved. RCD was found superior to BDR in terms of treatment response. For those who required 2nd line chemotherapy, the median time to next treatment was 22 months. To conclude, a late presentation and higher risk categories were common in our cohort of patients. Treatment outcome was comparable to those reported in western literature. RCD regimen was found to be a better treatment option in terms of overall survival.

Maternal and Fetal Outcomes of Pregnancy in Patients with Immune Thrombocytopenia.

INTRODUCTION: Immune thrombocytopenia (ITP) complicates 1-2 cases/10,000 pregnancies in India. Management of these patients is a challenge as it is associated with potential risks of maternal bleeding episodes and neonatal alloimmune thrombocytopenia (NAITP). OBJECTIVE: To study the maternal and fetal/neonatal outcome of pregnancy in Indian patients with ITP and identify the risk factors for NAITP. MATERIALS AND METHODS: In this retrospective study, all ITP patients with pregnancy who were diagnosed and treated at our center over 8 years (August 2010- August 2018) were evaluated for their hematological, obstetrical, and fetal outcomes. RESULTS: Twenty-nine pregnancies in 27 ITP patients were studied. The mean interval between the diagnosis of ITP and each pregnancy was 29 ± 14.9 months. The mean baseline platelet count was 0.18 ± 0.05 X 109/L. Twenty-seven (93.1%) cases were treated with oral prednisolone. Twenty deliveries (69.0%) were vaginal and 9 (31.0%) deliveries were by cesarean section. There were no major bleeding episodes during pregnancy or delivery.The mean neonatal platelet count was 1.23 ± 0.58 × 109/L at birth. NAITP was seen in 3 (3.5%) neonates. No bleeds or intracranial hemorrhages were observed. Only maternal platelet count < 50 X 109/L at delivery showed a statistical correlation with NAITP (p = 0.022). There was no positive correlation between NAITP and the duration of maternal ITP, the timing of ITP onset, or type of treatment. CONCLUSION: Successful outcome of pregnancies in ITP patients is possible, and the risk of maternal bleeding and NAITP is low.

Comparative evaluation of vestibular incision subperiosteal tunnel access with platelet-rich fibrin and connective tissue graft in the management of multiple gingival recession defects: A randomized clinical study.

BACKGROUND: The etiology of gingival recession is often multifactorial. Wide array of surgical techniques are available to manage gingival recession. The aim of the present study was to compare, minimally invasive approach (vestibular incision subperiosteal tunnel access [VISTA]), in combination with platelet-rich fibrin (PRF) and connective tissue graft (CTG) in the management of multiple recession defects in maxillary anterior region. MATERIALS AND METHODS: A total of 32 sites from 10 systemically healthy controls were allocated randomly to VISTA with PRF (VISTA + PRF) and VISTA with CTG (VISTA + CTG). Plaque index, gingival index, Probing probing pocket depth (PPD), relative attachment level (RAL), recession depth (RD), recession width (RW), width of keratinized gingiva (WKG), and percentage of root coverage (%RC) were calculated at 6 months postoperatively. RESULTS: Results showed significant improvement in mean PPD, RAL, RD, RW, and KTW. %RC in VISTA + PRF and VISTA + CTG was 83.25% ± 25.02% and 86.43% ± 22.79%, respectively, at 6 months. There were no significant differences in the parameters between the VISTA + PRF and VISTA + CTG groups. CONCLUSION: VISTA is a minimally invasive surgical approach, which can be combined with CTG or PRF in the management of Miller's Class I and Class II recession defects, with predictable outcomes. There were significant improvements in the clinical parameters from baseline to 6 months in both the groups. To match with the CTG, which is the gold standard procedure, PRF can be used as an alternative for treating multiple recession defects.

Clinicopathological and immunophenotypic features of early T cell precursor acute lymphoblastic leukaemia: A flow cytometry score for the initial diagnosis.

OBJECTIVE: To assess the prevalence of early T precursor-acute lymphoblastic leukaemia (ETP-ALL), study its clinicopathological features and devise a 'flow score' based on immunophenotypic profiles. MATERIAL METHODS: This was a retrospective study where clinical and laboratory data of all consecutive T-ALL cases were analysed to identify features differentiating ETP from non-ETP-ALL. The utility of a flow score based on the five commonly used markers in leukaemia panels for T-ALL (CD34, CD8, CD5, CD13 and CD33) was evaluated to differentiate ETP from non-ETP-ALL. RESULTS: Early T precursor-acute lymphoblastic leukaemia constituted 24.2% (n = 29) of all T-ALL cases. It was significantly more common in adults (30.2%) as compared to paediatric (17.5%) patients (P = .046). The median age of presentation was significantly higher than the non-ETP group. (24 vs 19 years; P = .01). Patients with ETP-ALL usually presented with organomegaly, lymphadenopathy, lower levels of haemoglobin, total leucocyte count, peripheral blood blast proportion and LDH levels as compared to non-ETP-ALL. The majority of ETP-ALL cases had L2 morphology with a moderate amount of cytoplasm showing frequent blebbing. A flow score cut-off value of ≥3 on ROC curve analysis had a sensitivity and specificity of 100% and 94.6% respectively. CONCLUSION: Early T precursor-acute lymphoblastic leukaemia had unique clinical and laboratory features. The prevalence of this entity is more common in the adult population. A flow score based on a minimum of five widely used markers can confidently identify ETP-ALL and should be included in the primary panel of markers used for flow cytometric analysis.

Pregnancy and Its Successful Outcome in a Patient with Multiple Myeloma.

Multiple myeloma is a B-cell neoplastic disorder and represents 1% of all cancers and 13% of hematological malignancies. It is predominantly a disease of elderly, and less than 3% of all cases are below the age of 40 years. We present the case of a 29-year-old lady with multiple myeloma who had spontaneous conception during maintenance therapy and subsequently a successful pregnancy outcome. She gave birth to a healthy female infant through normal vaginal delivery and subsequently could remain off therapy for 5 years. Since the patient had a history of abortions and stillbirth, it was a precious pregnancy and we could successfully salvage both the mother and the baby. Young female patients of myeloma who are in remission can be encouraged to start a family during their reproductive years with the support of a comprehensive care team of hematologists/oncologists and obstetricians.

Mycotic Aneurysm of Brachial Artery in Case of Aplastic Anemia.

Pseudo-aneurysm of the brachial artery is relatively rare condition affecting the arterial vessels of the limbs. It can be due to trauma, infections or systemic vasculitis. We report a patient with aplastic anemia who developed pseudoaneurysm of the brachial artery following an episode of bacterial sepsis. Methicillin resistant Staphylococcus aureus (MRSA) was isolated on blood culture. Patient was treated with systemic antibiotics and underwent embolization of the pseudo-aneurysm. He later developed vascular insufficiency of the forearm and a stent had to be placed in the brachial artery at the site of calcified thrombus inside the pseudo-aneurysm to produce the patency of the artery and ensure adequate blood flow to forearm and hand.

Herbal derivatives in the management of mouth opening in oral submucous fibrosis-A network meta-analysis.

OBJECTIVES: Oral submucous fibrosis is a potentially malignant disorder commonly seen in Asian countries. In this disease, the mucosa becomes stiff and patients have difficulty in mouth opening, thus affecting their quality of life. Recently, practitioners are focusing on herbal derivatives instead of commonly practiced intralesional steroids for the management of this disorder. Hence, we conducted a network meta-analysis of randomized clinical trials on herbal derivatives to identify the most effective treatment for oral submucous fibrosis. MATERIALS AND METHODS: Electronic search for articles published in various journals was undertaken through various search engines till January 2020. 14 articles were selected which had performed randomized control trials of herbal derivatives against control/placebo. Frequentist network meta-analysis was performed using R studio software, and effective treatment ranking was derived. RESULTS: The treatment ranking was generated, and relative to mouth opening, the most effective treatment was lycopene administered along with vitamin E and the second effective drug was aloe vera gel. CONCLUSION: This network meta-analysis highlights the efficacy of some of the drugs of herbal origin which can be implemented by the practitioners in the management of initial stages of oral submucous fibrosis.

A case of compound heterozygous hemoglobin Köln/hemoglobin E in an Indian family.

Hemoglobin Köln, is the most common unstable hemoglobin variant worldwide, yet has only rarely been reported in Indians. Herein we report a case of coinheritance of Hb Köln and Hb E, which to the best of our knowledge has not been reported in the literature so far. The patient presented with mild symptoms of hemolysis with no previous history of blood transfusions.