Peripheral Blood Nucleated Cells Research Articles

Exagamglogene Autotemcel for Severe Sickle Cell Disease

Background: Exagamglogene autotemcel (exa-cel) is a non-viral cell therapy designed to reactivate fetal hemoglobin via ex vivo CRISPR-Cas9 gene-editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of the BCL11A gene in patients (pts) with severe sickle cell disease (SCD). We report that in a pre-specified interim analysis, the pivotal CLIMB SCD-121 trial of exa-cel met primary and key secondary endpoints. Methods: CLIMB SCD-121 is an ongoing, 24-mo, phase 3 trial of exa-cel in pts age 12-35y with SCD and a history of ≥2 VOCs/y in 2y prior to screening. Primary efficacy endpoint is proportion of pts free of severe VOCs for ≥12 consecutive months (mos) (VF12); key secondary efficacy endpoints are proportion of pts free from inpatient hospitalization for severe VOCs for ≥12 consecutive mos (HF12) and proportion of pts free from severe VOCs for ≥9 consecutive mos (VF9). Evaluable pts for VF12 and HF12 had ≥16 mos follow-up after exa-cel infusion; pts evaluable for VF9 had ≥12 mos follow-up after infusion. Evaluation of primary and key secondary endpoints began 60 days after last RBC transfusion for post-transplant support or SCD management. Pts completing trial enrolled in long-term follow-up Study 131. Mean (SD) are shown except where noted. Results: As of 10 Feb 2023, 42 pts with SCD (age 21.2[range 12-34]y; 12[28.6%] age ≥12 to <18y; 4.2 VOCs/y at baseline) received exa-cel. Following infusion, all pts engrafted neutrophils and platelets (median 27 and 34.5 days, respectively). 19/20 (95.0%) pts evaluable for primary endpoint were free of VOCs for ≥12 consecutive mos (VF12; 95% CI, 75.1% to 99.9%; P<0.0001), 20/20 (100%) were free from hospitalizations for VOCs for ≥12 consecutive mos (HF12; 95% CI, 83.2 to 100.0; P<0.0001), and 29/30 (96.7%) were free of VOCs for ≥9 consecutive mos (VF9; 95% CI, 82.8 to 99.9; P<0.0001). In pts achieving VF12, VOC free duration was 21.8 (range 12.3-41.4) mos; 18 pts remained VOC free through follow-up and 1 pt had an adjudicated VOC in the setting of parvovirus infection ~22.8 mos after exa-cel; pt recovered fully and has since been VOC free (Fig). For all pts, total Hb was 12.1 g/dL at Month 3 and was maintained at ≥11.0 g/dL from Month 6 onward; HbF was 36.0% at Month 3 and was generally maintained at ≥ 40.0% from Month 6 onward with pancellular distribution (≥95% RBCs expressing HbF). Proportion of edited BCL11A alleles was stable over time in bone marrow CD34 + and peripheral blood nucleated cells. 36/39 pts with ≥60 days follow-up after last RBC transfusion (including those not yet evaluable) remained VOC free (up to 41.4 mos; Fig). Quality-of-life (QOL) measures showed clinically significant improvements from baseline. All pts had ≥1 adverse event (AE), most were Grade 1 or 2; 40 (95.2%) pts had AEs of Grade 3 or 4 severity. Most common AEs were nausea (66.7%), stomatitis (61.9%), febrile neutropenia (52.4%), headache (52.4%), and vomiting (52.4%). Most AEs and serious AEs (SAEs) occurred within first 6 mos after infusion. No pts had SAEs considered related to exa-cel. As previously reported, 1 pt died from respiratory failure due to COVID-19 unrelated to exa-cel. There were no study discontinuations or malignancies. Conclusions: The CLIMB SCD-121 trial met primary and key secondary endpoints, with exa-cel treatment resulting in early and sustained increases in Hb and HbF leading to elimination of VOCs in 95% of pts, elimination of inpatient hospitalization for VOCs in 100% of pts and improved QOL. Safety profile of exa-cel was generally consistent with myeloablative busulfan conditioning and autologous transplantation. These results show exa-cel has the potential to deliver a one-time functional cure to pts with severe SCD.

Exagamglogene Autotemcel for Transfusion-Dependent β-Thalassemia

Background: Exagamglogene autotemcel (exa-cel) is a non-viral cell therapy designed to reactivate fetal hemoglobin (HbF) via ex vivo CRISPR-Cas9 gene-editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A in patients (pts) with transfusion-dependent β-thalassemia (TDT). Here we report that in a pre-specified interim analysis, the pivotal CLIMB THAL-111 trial of exa-cel met primary and key secondary endpoints. Methods: CLIMB THAL-111 is an ongoing, 24-month (mo), phase 3 trial of exa-cel in pts age 12-35y with TDT and a history of ≥100 mL/kg/y or ≥10 U/y packed red blood cell (RBC) transfusions in the 2y before screening. Primary endpoint is transfusion independence defined as proportion of pts maintaining a weighted average hemoglobin (Hb) ≥9 g/dL without RBC transfusion for ≥12 consecutive mos (TI12). Key secondary endpoint is proportion of pts maintaining a weighted average Hb ≥9 g/dL without RBC transfusion for ≥6 consecutive mos (TI6). Evaluable pts had ≥16 mos of follow-up after exa-cel infusion. Evaluation of TI12 and TI6 started 60 days after last RBC transfusion for post-transplant support or TDT management. Pts completing trial enrolled in long-term follow-up Study 131. Mean (SD) shown except where noted. Results: As of 16 Jan 2023, 52 pts (mean age 21.5[range 12-35]y; 18[34.6%] age ≥12 to <18y; 31[59.6%] with severe genotypes [β 0/β 0 or β 0/β 0-like], median annualized transfusion volume 201.0 mL/kg) received exa-cel; median follow-up 20.4 (range 2.1-48.1) mos. Following infusion, all pts engrafted neutrophils and platelets (median 29 and 44 days, respectively). Of the 35 pts evaluable for primary and key secondary endpoints, 32 (91.4%) achieved TI12 and TI6 (95% CI: 76.9%, 98.2%; P<0.0001). Pts achieving TI12 stopped transfusions 35.2 (SD, 18.5) days after exa-cel infusion and remained transfusion independent for 22.5 (range, 13.3, 45.1) mos (Fig). For 3 pts not achieving TI12, one had reductions in annualized RBC transfusion volume of 83.9%, while the others have been transfusion-free for 7.3 mos and 4.0 mos starting 60 days after the last transfusion. For all pts, total Hb was 11.4 g/dL at Month 3 (≥12g/dL Month 6 onward) and HbF was 7.7 g/dL at Month 3 (≥ 10 g/dL Month 6 onward) with pancellular distribution (≥95% RBCs expressing HbF Month 6 onward). Proportion of edited BCL11A alleles was stable over time in bone marrow CD34 + and peripheral blood nucleated cells. Pts not yet evaluable and with sufficient follow-up were also transfusion-free. Quality-of-life (QOL) measures showed clinically significant improvements. All pts had ≥1 adverse event (AE), most were Grade 1 or 2; 46 (88.5%) pts had AEs of Grade 3 or 4 severity. Most common AEs were febrile neutropenia (61.5%), headache (53.8%), and stomatitis (50.0%). Most AEs and serious AEs (SAEs) occurred within first 6 mos after infusion. Two pts had SAEs considered related to exa-cel: headache, hemophagocytic lymphohistiocytosis (HLH), acute respiratory distress syndrome and idiopathic pneumonia syndrome (latter also considered related to busulfan) all in the context of HLH (n=1) and delayed engraftment and thrombocytopenia (both also considered related to busulfan) (n=1), which all resolved. There were no deaths, discontinuations, or malignancies. Conclusions: The CLIMB THAL-111 trial met primary and key secondary endpoints, with exa-cel treatment resulting in early and sustained increases in Hb and HbF leading to transfusion independence in >90% of pts with TDT and improved QOL. Safety profile of exa-cel was generally consistent with myeloablative busulfan conditioning and autologous transplantation. These results show exa-cel has the potential to deliver a one-time functional cure to pts with TDT.

Fixed-Duration Pirtobrutinib Combined with Venetoclax ± Rituximab in Relapsed/Refractory Chronic Lymphocytic Leukemia: Updated Results, Including MRD Data, from the BRUIN Phase 1b Study

Background: Covalent (c) Bruton tyrosine kinase inhibitors (BTKi) have transformed the management of chronic lymphocytic leukemia (CLL). Recent clinical studies have evaluated the safety and efficacy of fixed-duration regimens with venetoclax and cBTKi. While these combinations are effective, their use may be limited by toxicity. Pirtobrutinib, a highly selective, non-covalent (reversible) BTKi, has shown promising safety and efficacy in heavily pretreated relapsed or refractory (R/R) CLL patients (pts). Here, we report the safety and efficacy of fixed-duration pirtobrutinib combined with venetoclax ± rituximab in pts with R/R CLL. Methods: Pts with R/R CLL were eligible for pirtobrutinib combined with venetoclax ± rituximab in the phase 1b portion of the multicenter phase 1/2 BRUIN study (NCT03740529). Prior cBTKi therapy was allowed, but not prior venetoclax. Pts were enrolled into sequential cohorts, first in the pirtobrutinib + venetoclax (PV) cohort and then the PV + rituximab (PVR) cohort. Pirtobrutinib 200 mg QD was started on cycle 1 day 1, and venetoclax was started on cycle 2 day 1 with the standard 5-week dose ramp to 400 mg QD target dose. PV was given for up to 24 cycles; each cycle was 28 days. For the PVR cohort, rituximab was given at 375 mg/m 2 on cycle 1 day 1 and then 500 mg/m 2 on day 1 of cycles 2-6. The primary endpoint was safety as assessed by treatment-emergent adverse events (TEAEs) graded according to CTCAE v5.0. Other key endpoints included overall response rate (ORR), progression-free survival (PFS), and minimal residual disease (MRD). Undetectable MRD (uMRD) was defined as less than 1 CLL cell/10,000 nucleated cells (<1x10 -4) in peripheral blood by ClonoSEQ® assay (Adaptive Biotechnologies, Seattle, WA). A data cut of May 5, 2023, was utilized. Results: Fifteen pts received PV, and 10 received PVR. Median age was 66 years (range, 49-77) for those who received PV and 69 (range, 39-78) for those who received PVR. Median prior lines of therapy was 1 (range, 1-2) for the PV cohort and 2 (range 1-4) for the PVR cohort. Most pts had received prior chemotherapy (PV=53%; PVR=60%), CD20 monoclonal antibody (73%; 70%), and cBTKi (73%; 60%). The majority of pts had IGHV unmutated CLL (PV=73%; PVR=89%). ORR was 93.3% (95% CI, 68.1-99.8) for the 15 pts receiving PV and 100% (95% CI, 69.2-100.0) for the 10 pts receiving PVR, with 10 complete responses (PV=7; PVR=3). Median duration of follow-up for PFS was 22.1 months (IQR, 20.1-25.7) for PV and 22.1 months (IQR, 20.7-22.1) for PVR; PFS at 18 months was 92.9% (95% CI, 59.1-99.0) for the 15 pts receiving PV and 80.0% (95% CI, 40.9-94.6) for the 10 pts receiving PVR. The overall uMRD rate at cycle 13 was 70.8% (PV=10; PVR=7) for the 24 evaluable pts, with 87.5% (PV=12; PVR=9) of pts achieving uMRD at some time during the trial and all but one pt sustaining uMRD during subsequent MRD assessments ( Figure). Twenty-two pts discontinued treatment (PV=14; PVR=8); of these, 14 completed all 24 cycles of therapy (PV=9; PVR=5) and 8 discontinued early because of progressive disease (PV=2), adverse events (PVR=2), protocol noncompliance (PV=1), death unrelated to treatment (PVR=1), or other reasons (PV=2). Three pts (PV=1; PVR=2) continue to receive treatment. The median relative dose intensity was similar in both cohorts (PV=97.9%; PVR=98.6%). The most common TEAE of any grade included nausea (PV=60.0%; PVR=40.0%), fatigue (53.3%; 50.0%), and diarrhea (46.7%; 60.0%). The most common grade ≥3 TEAE was neutropenia/neutrophil count decreased (PV=46.7%; PVR=60.0%). Grade ≥3 clinical tumor lysis syndrome occurred during venetoclax dose escalation (PV=2), including a grade 3 case that resolved spontaneously after 24 hours and a grade 4 case that resolved after short-term intravenous fluids. Treatment-related adverse events led to dose reductions in three pts (PV=1; PVR=2) and treatment discontinuation in two pts (PVR=2). Conclusions: Fixed-duration pirtobrutinib combined with venetoclax ± rituximab was well tolerated and demonstrated sufficiently promising efficacy to warrant further investigation in pts with R/R CLL. The BRUIN CLL-322 phase 3 trial comparing PVR vs VR in previously treated CLL is currently enrolling pts (NCT04965493).

Identification of potential hub genes linked to immune and metabolic alterations in postoperative systemic inflammatory dysregulation.

Postoperative systemic inflammatory dysregulation (PSID) is characterised by strongly interlinked immune and metabolic abnormalities. However, the hub genes responsible for the interconnections between these two systemic alterations remain to be identified. We analysed differentially expressed genes (DEGs) of individual peripheral blood nucleated cells in patients with PSID (n = 21, CRP > 250 mg/L) and control patients (n = 25, CRP < 75 mg/L) following major abdominal surgery, along with their biological functions. Correlation analyses were conducted to explore the interconnections of immune-related DEGs (irDEGs) and metabolism-related DEGs (mrDEGs). Two methods were used to screen hub genes for irDEGs and mrDEGs: we screened for hub genes among DEGs via 12 algorithms using CytoHubba in Cytoscape, and also screened for hub immune-related and metabolic-related genes using weighted gene co-expression network analysis. The hub genes selected were involved in the interaction between changes in immunity and metabolism in PSID. Finally, we validated our results in mice with PSID to confirm the findings. We identified 512 upregulated and 254 downregulated DEGs in patients with PSID compared with controls. Gene enrichment analysis revealed that DEGs were significantly associated with immune- and metabolism-related biological processes and pathways. Correlation analyses revealed a close association between irDEGs and mrDEGs. Fourteen unique hub genes were identified via 12 screening algorithms using CytoHubba in Cytoscape and via weighted gene co-expression network analysis. Among these, CD28, CD40LG, MAPK14, and S100A12 were identified as hub genes among both immune- and metabolism-related genes; these genes play a critical role in the interaction between alterations in immunity and metabolism in PSID. The experimental results also showed that the expression of these genes was significantly altered in PSID mice. This study identified hub genes associated with immune and metabolic alterations in patients with PSID and hub genes that link these alterations. These findings provide novel insights into the mechanisms underlying immune and metabolic interactions and new targets for clinical treatment can be proposed on this basis.

Fetal nucleated red blood cells in maternal peripheral blood for non-invasive prenatal diagnosis

Fetal nucleated red blood cells in maternal peripheral blood for non-invasive prenatal diagnosis

Analytical errors in nucleated red blood cell enumeration.

Enumeration of nucleated red blood cells (NRBCs) in peripheral blood of dogs and cats is performed by manual counting during blood film evaluation. Automated methods have increased precision and accuracy; however, most analyzers cannot distinguish leukocytes and NRBCs. The Sysmex XN-V Series may distinguish NRBCs and leukocytes; however, analytical errors occur. We aimed to investigate cases with discrepant automated and manual NRBC counts, and to evaluate reasons for analytical errors. Data from samples with increased NRBCs were collected retrospectively and compared with manual counts performed on blood films using Spearman's correlation, Passing-Bablok agreement analysis, and Bland-Altman comparisons. Precision of the automated method and interobserver agreement of manual counts were evaluated. Cases with discrepant results were investigated. Agreement between the methods was good at ≤1NRBC ×109 /L in dogs and cats, and inadequate at ≥1NRBC ×109 /L. The automated method demonstrated a negative proportional difference to the manual method. Precision was good for the automated method (overall CV 7.1%) and interobserver agreement for the manual method was poor overall (mean CV 27.3%, range 0%-106.1%). Inaccuracies in NRBC enumeration by the automated method occurred with high hematocrits, the mergence of the cell fragments and leukocyte clouds, and the presence of earlier erythroid precursors. NRBC enumeration by the WNR channel on the Sysmex XN-1000 V is precise and has good agreement with manual counts in canine and feline blood samples at ≤1NRBC ×109 /L. Manual film review is indicated for samples with ≥1NRBC ×109 /L, earlier erythroid precursors, samples from greyhounds and dehydrated patients, and if gating errors are noted.

The Impact of Different Types of Physical Effort on the Expression of Selected Chemokine and Interleukin Receptor Genes in Peripheral Blood Cells.

This study aimed to assess the post-effort transcriptional changes of selected genes encoding receptors for chemokines and interleukins in young, physically active men to better understand the immunomodulatory effect of physical activity. The participants, aged 16-21 years, performed physical exercise tasks of either a maximal multistage 20 m shuttle-run test (beep test) or a repeated speed ability test. The expression of selected genes encoding receptors for chemokines and interleukins in nucleated peripheral blood cells was determined using RT-qPCR. Aerobic endurance activity was a positive stimulant that induced increased expression of CCR1 and CCR2 genes following lactate recovery, while the maximum expression of CCR5 was found immediately post-effort. The increase in the expression of inflammation-related genes encoding chemokine receptors triggered by aerobic effort strengthens the theory that physical effort induces sterile inflammation. Different profiles of studied chemokine receptor gene expression induced by short-term anaerobic effort suggest that not all types of physical effort activate the same immunological pathways. A significant increase in IL17RA gene expression after the beep test confirmed the hypothesis that cells expressing this receptor, including Th17 lymphocyte subsets, can be involved in the creation of an immune response after endurance efforts.

Integrated strategy for biomarkers of stable coronary heart disease with phlegm and blood stasis syndrome based on RNA-seq and network pharmacology

This study aimed to analyze the biological foundation and biomarkers of stable coronary heart disease(CHD) with phlegm and blood stasis(PBS) syndrome based on RNA-seq and network pharmacology. Peripheral blood nucleated cells from five CHD patients with PBS syndrome, five CHD patients with non-PBS syndrome, and five healthy adults were collected for RNA-seq. The specific targets of CHD with PBS syndrome were determined by differential gene expression analysis and Venn diagram analysis. The active ingredients of Danlou Tablets were screened out from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the &quot;component-target&quot; prediction was completed through PubChem and SwissTargetPrediction. The &quot;drug-ingredient-target-signaling pathway&quot; network of Danlou Tablets against CHD with PBS syndrome was optimized by Cytoscape software. After the target biomarkers were identified, 90 participants were enrolled for diagnostic tests, and 30 CHD patients with PBS syndrome were included in before-and-after experiment to determine the therapeutic effect of Danlou Tablets on those targets. As revealed by RNA-seq and Venn diagram analysis, 200 specific genes were identified for CHD with PBS syndrome. A total of 1 118 potential therapeutic targets of Danlou Tablets were predicted through network pharmacology. Through integrated analysis of the two gene sets, 13 key targets of Danlou Tablets in the treatment of CHD with PBS syndrome were screened out, including CSF1, AKR1C2, PDGFRB, ARG1, CNR2, ALOX15B, ALDH1A1, CTSL, PLA2G7, LAP3, AKR1C3, IGFBP3, and CA1. They were presumably the biomarkers of CHD with PBS syndrome. The ELISA test further showed that CSF1 was significantly up-regulated in the peripheral blood of CHD patients with PBS syndrome, and was significantly down-regulated after Danlou Tablets intervention. CSF1 may be a biomarker for CHD with PBS syndrome, and it is positively correlated with the severity of the disease. The diagnostic cut-off of CSF1 for CHD with PBS syndrome was 286 pg·mL~(-1).

Transamniotic Fetal Administration of Genetically Modified Hematopoietic Stem Cells Carrying a Human Transgene in a Syngeneic Rat Model.

Hematopoietic stem cell (HSC)-based gene therapy has already reached clinical reality in a few applications. Fetal administration of genetically modified HSCs has only been feasible to date through invasive and morbid methods. It has been recently shown that native donor HSCs can reach the fetal circulation and bone marrow after simple delivery into the amniotic fluid, at least in a syngeneic healthy model. We sought to determine whether the transamniotic route could also be a practical alternative for the fetal administration of genetically modified HSCs in a comparable model. Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all their fetuses (n = 47) on gestational day 17 (E17; term = E21-22) of donor HSCs genetically modified using a custom lentiviral vector designed to constitutively express both a firefly luciferase reporter gene and a human adenosine deaminase (ADA) transgene. Donor HSCs consisted of syngeneic cells isolated from the amniotic fluid and phenotyped by flow cytometry. Fetuses were euthanized at term, when seven select sites relevant to HSC-based therapies were screened for either luciferase activity by luminometry or for the presence of human ADA mRNA by digital droplet polymerase chain reaction (ddPCR). Among survivors (30/47; 64%), positive luminescence and positive human ADA expression were detected in the bone marrow (respectively, 33% and 76%), liver (respectively, 11% and 81%), spleen (respectively, 11% and 67%), thymus (respectively, 33% and 67%), lungs (respectively, 44% and 86%), and brain (respectively, 22% and 90%). Nucleated peripheral blood cells were analyzed only by ddPCR, showing positive human ADA expression at 54%. We conclude that genetically modified HSCs can reach the fetal circulation and fetal bone marrow after simple intra-amniotic administration in a syngeneic rat model. Gene therapy by transamniotic HSC delivery may become a practicable, minimally invasive strategy for the prenatal treatment of select hemoglobinopathies, immunodeficiencies, and inherited metabolic disorders.

Relationship between glutathione S-transferase M1 gene polymorphism and gastric cancer

This experiment was designed to investigate the relationship between glutathione S-transferase M1 (GSTM1) gene polymorphism and gastric cancer (GC). For this purpose, from January 2013 to December 2014, 116 patients with GC diagnosed in the Department of Gastroenterology of our hospital and 120 healthy people in the physical examination center were selected as the research objects. 116 patients with GC served as the observation group and 120 healthy people in the physical examination center served as the control group. Collect and isolate the peripheral blood nucleated cells of the subjects, obtain the GSTM1 gene polymorphism by sequencing, analyze the differences of GSTM1 genotype between the two groups, compare the differences of clinicopathological characteristics of patients with different genotypes in the observation group, look for the survival relative risk factors of patients with GC, and analyze the risk factors of death risk of GC by multivariate Cox risk proportional regression. Results showed that the proportion of GSTM1 (-) in the observation group (62.07%) was raised compared with the control group (48.33%) (p<0.05). There was a correlation between GSTM1 gene polymorphism and smoking, TNM stage differentiation and GSTM1 gene polymorphism in the observation group. The specific analysis found that the proportion of non-smoking, stage I-III and low differentiation in the GSTM1 (-) group was raised compared with that in the GSTM1 (+) group (p<0.05). TNM stage, differentiation degree and GSTM1 gene polymorphism were correlated with the median survival time of patients with GC (p<0.05). Further multivariate Cox risk proportional regression analysis showed that TNM stage IV, low differentiation and GSTM1 (-) the relative risk coefficients of death in patients with GC were stage Ⅰ - Ⅲ, high/medium differentiation and GSTM1, respectively (+) patients were 1.75, 1.46, and 2.14 times higher. In conclusion, GSTM1 gene polymorphism is associated with susceptibility to GC, and the GSTM1 deletion genotype is an unfavourable factor for poor prognosis in patients with GC.

Study of Hemato-morphological Features in Neuroblastoma Infiltrating Marrow.

Objective Neuroblastoma typically affects children within the first 5 years of life and accounts for 10% of all pediatric malignancies. Neuroblastoma at onset may manifest as a localized or metastatic illness. The aim of this study was to identify hematomorphological features in neuroblastoma infiltrating marrow as well as to ascertain the prevalence of bone marrow infiltration in neuroblastoma. Materials and Methods This retrospective study included newly diagnosed 79 cases of neuroblastoma, which were referred for bone marrow examination for the staging of the disease. Medical records were retrieved to acquire hematomorphological findings of peripheral blood and bone marrow smears. Statistical Package for Social Sciences, IBM Inc., USA, version 21.0 was used to analyze the data. Results The interquartile age range of neuroblastoma cases was 24.0 to 72.0 months (median = 48 months) with a male to female ratio of 2.7:1. Also, 55.6% (44/79) of cases in the study population showed evidence of marrow infiltration. The bone marrow infiltration was significantly linked to thrombocytopenia ( p = 0.043) and nucleated red blood cells ( p = 0.003) in peripheral blood. The bone marrow smears of cases with infiltration showed a significant shift to the left in the myeloid series ( p = 0.001) and an increased number of erythroid cells ( p = 0.001). Conclusion For neuroblastoma patients, a diligent, exhaustive search for infiltrating cells in bone marrow is advised if thrombocytopenia or nucleated red blood cells are identified on a peripheral blood smear and bone marrow smears showed myeloid left shift with an increased number of erythroid cells.

Single-cell transcriptome profiling reveals heterogeneous neutrophils with prognostic values in sepsis

SummaryNeutrophils constitute the largest proportion of nucleated peripheral blood cells, and neutrophils have substantial heterogeneity. We profiled nearly 300,000 human peripheral blood cells in this study using single-cell RNA sequencing. A large proportion (>50%) of these cells were annotated as neutrophils. Neutrophils were further clustered into four subtypes, including Neu1, Neu2, Neu3, and Neu4. Neu1 is characterized by high expression of MMP9, HP, and RGL4. Neu1 was associated with septic shock and significantly correlated with the sequential organ failure assessment (SOFA) score. A gene expression module in Neu1 named Neu1_C (characterized by expression of NFKBIA, CXCL8, G0S2, and FTH1) was highly predictive of septic shock with an area under the curve of 0.81. The results were extensively validated in external bulk datasets by using single-cell deconvolution methods. In summary, our study establishes a general framework for studying neutrophil-related mechanisms, prognostic biomarkers, and potential therapeutic targets for septic shock.

The Cytogenetic Profile of Primary and Secondary Plasma Cell Leukemia: Etiopathogenetic Perspectives, Prognostic Impact and Clinical Relevance to Newly Diagnosed Multiple Myeloma with Differential Circulating Clonal Plasma Cells.

Plasma cell leukemia (PCL) is a rare and aggressive plasma cell dyscrasia that may appear as de-novo leukemia (pPCL) or on the basis of a pre-existing multiple myeloma (MM), called secondary plasma cell leukemia (sPCL). In this prospective study, we have applied a broad panel of FISH probes in 965 newly diagnosed MM (NDMM) and 44 PCL cases of both types to reveal the particular cytogenetic differences among the three plasma cell dyscrasias. In order to evaluate the frequency and patterns of clonal evolution, the same FISH panel was applied both at diagnosis and at the time of first relapse for 81 relapsed MM patients and both at MM diagnosis and during sPCL transformation for the 19 sPCL cases described here. pPCL was characterized by frequent MYC translocations and t(11;14) with a 11q13 breakpoint centered on the MYEOV gene, not commonly seen in MM. sPCL had a higher number of FISH abnormalities and was strongly associated with the presence of del(17p13), either acquired at the initial MM stage or as a newly acquired lesion upon leukemogenesis in the context of the apparent clonal evolution observed in sPCL. In clinical terms, sPCL showed a shorter overall survival than pPCL with either standard or high-risk (t(4;14) and/or t(14;16) and/or del(17p13) and/or ≥3 concomitant aberrations) abnormalities (median 5 months vs. 21 and 11 months respectively, p < 0.001), suggesting a prognostic stratification based on cytogenetic background. These observations proved relevant in the NDMM setting, where higher levels of circulating plasma cells (CPCs) were strongly associated with high-risk cytogenetics (median frequency of CPCs: 0.11% of peripheral blood nucleated cells for high-risk vs. 0.007% for standard-risk NDMM, p < 0.0001). Most importantly, the combined evaluation of CPCs (higher or lower than a cut-off of 0.03%), together with patients’ cytogenetic status, could be used for an improved prognostic stratification of NDMM patients.

Artificial intelligence-based classification of peripheral blood nucleated cells using label-free imaging flow cytometry.

Label-free image identification of circulating rare cells, such as circulating tumor cells within peripheral blood nucleated cells (PBNCs), the vast majority of which are white blood cells (WBCs), remains challenging. We previously described developing label-free image cytometry for classifying live cells using computer vision technology for pattern recognition, based on the subcellular structure of the quantitative phase microscopy images. We applied our image recognition methods to cells flowing in a flow cytometer microfluidic channel, and differentiated WBCs from cancer cell lines (area under receiver operating characteristic curve = 0.957). We then applied this method to healthy volunteers' and advanced cancer patients' blood samples and found that the non-WBC fraction rates (NWBC-FRs), defined as the percentage of cells classified as non-WBCs of the total PBNCs, were significantly higher in cancer patients than in healthy volunteers. Furthermore, we monitored NWBC-FRs over the therapeutic courses in cancer patients, which revealed the potential ability in monitoring the clinical status during therapy. Our image recognition system has the potential to provide a morphological diagnostic tool for circulating rare cells as non-WBC fractions.

Correlations of FUT3 gene polymorphisms with colon polyps

ABSTRACT Objective: To explore the relationship between fut3 gene polymorphism and colonic polyps. Methods: Two hundred patients with colonic polyps and 200 healthy people in our hospital in recent 3 years were taken as the research objects, as the disease group and the control group, respectively. The disease group was divided into cancerous colonic polyps group (n = 50) and non-cancerous colonic polyps group (n = 150). The peripheral blood nucleated cells of the subjects were collected and isolated. The fut3 gene polymorphism was obtained by sequencing and analyzed combined with the expression of fut3 gene and the level of tumor markers. Results: The frequency of allele C at rs2561796 locus in the disease group was significantly higher than that in the control group (P < 0.05). The frequency of Ag genotype at rs441158 locus in the disease group was significantly higher than that in the control group, and the frequency of Ca genotype at rs2561796 locus was significantly lower than that in the control group (P < 0.05). In the disease group, the frequency of AA + Ag in the dominant model at rs441158 was significantly higher than that in the control group, and the frequency of Ca + AA in the invisible model at rs2561796 was significantly higher than that in the control group (P < 0.05). The frequency of CGC haplotype in the disease group was higher than that in the control group (P < 0.05). The linkage disequilibrium of rs441158 and rs2561796 loci of fut3 gene was high (d‘ = 0.423). The genotype of rs372725 of fut3 gene was correlated with the expression of fut3 gene (P < 0.05). The expression of fut3 gene in patients with CC genotype was significantly higher than that in patients with other genotypes (P < 0.05). Conclusion: fut3 gene polymorphism is associated with the susceptibility and carcinogenesis of colonic polyps.

Cord blood and peripheral nucleated red blood cells levels in term asphyxiated and non-asphyxiated new borns and neonatal outcome: a comparative study

Background: Perinatal asphyxia can cause severe hypoxic-ischemic damages in the organs and causes severe long-term consequences. It has recently been suggested that the increase in NRBC count in the umbilical vein of newborns can be considered a sign of birth asphyxia. The aim of the study was to evaluate the role of cord blood nucleated RBC count as a marker for presence and severity of birth asphyxia.Methods: This prospective observational comparative study was conducted among 30 asphyxiated (case) and 30 non-asphyxiated newborns (control) in Swami Dayanand Hospital. Number of nucleated red blood cells in cord blood and peripheral blood were counted per 100 leukocytes in smears in cases and control. Maternal parameters and neonate characteristics were recorded in a proforma.Results: NRBC count increased in cord blood during the first few hours of life in perinatal asphyxia (10.79±9.58) as compared with the healthy control subjects (4.03±2.87, p=0.002). Significant positive correlation of C-NRBC and P-NRBC with NICU stay (r=0.921, p&lt;0.0001 and r=0.698, p&lt;0.0001 respectively). Nucleated RBC count more than 18 can be used as predictor of abnormal neurological outcome with sensitivity 100% and specificity of 93.1%. The cord blood NRBC &gt;3/100 WBC has sensitivity of 92% and specificity of 77.8% in predicting birth asphyxia.Conclusions: Nucleated RBC count can be used as surrogate marker for birth asphyxia. It has significant positive correlation with severity of hypoxic ischemic encephalopathy, duration of NICU stay and duration of O2 requirement.

DNA Methylation Differences in Peripheral Blood of Patients with Anaphylaxis.

Objective To study the DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and to provide a new research direction and basis for the forensic diagnosis of shock caused by drug hypersensitiveness. Methods Methylation microarray was used to detect DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and normal subjects. Sequencing data and chip data were analyzed for differences in DNA methylation using R language methylkit, ChAMP package. Random forest algorithm was used to evaluate the importance of the DNA methylation differential sites. Results Differential sites of DNA methylation highly associated with anaphylaxis caused by cephalosporin drugs were obtained at loci such as ETS1, PRR23B and GNAS. Conclusion Cephalosporin allergy is associated with DNA methylation, and DNA methylation may be a new strategy for forensic identification of anaphylactic shock and death.

A Flow-Cytometric Analysis of Spectrum of Acute Myeloid Leukemia at Diagnosis

Background: Acute Myeloid Lymphoma is the clonal proliferation of non-lymphoid blasts comprising at least 20% of total nucleated cells either in bone marrow or peripheral blood. In the recent years, flow cytometry has emerged as a powerful diagnostic tool for AML due to its impact on treatment and prognosis.&#x0D; Aims &amp; Objectives:&#x0D; &#x0D; To analyse the flow cytometry findings in patients diagnosed as acute myeloid leukemias.&#x0D; To evaluate variations in flow cytometry expression in various subtypes of acute myeloid leukemia&#x0D; &#x0D; Materials &amp; Methods: Patients diagnosed as acute myeloid leukaemia on peripheral smears were subjected to flow cytometry analysis. This was a four-year study from July 2015 to June 2017 retrospectively and from July 2017 to June 2019 prospectively.&#x0D; Results: A total of 27 cases diagnosed as Acute Myeloid Leukemia (AML) were included in the study. Acute Promyelocytic Leukemia was observed to be the most common subtype. The most commonly expressed myeloid antigens were CD13 and CD33. There was an aberrant expression of CD7 and CD56 in 1 case each indicating adverse prognosis.&#x0D; Conclusion: Immunophenotyping of the myeloid cells by flow cytometry has revolutionised the diagnosis of acute myeloid leukemias. It aids in confirming the morphological diagnosis, and also helps in assigning specific lineage, accurate sub classification and adequate treatment in challenging cases. Aberrant expressions were observed in 3 cases of AML. Aberrant antigen expression is associated with a poor outcome. Flow cytometry results interpreted with morphology are not only complementary but also conclusive aiding in therapeutics and predicting prognosis.

Enhanced Isolation of Fetal Nucleated Red Blood Cells by Enythrocyte-Leukocyte Hybrid Membrane-Coated Magnetic Nanoparticles for Noninvasive Pregnant Diagnostics.

Fetal nucleated red blood cells (fNRBCs) in maternal peripheral blood containing the whole genetic information of the fetus may serve for noninvasive pregnant diagnostics (NIPD). However, the fetal cell-based NIPD is seriously limited by the poor purity of the isolated fNRBCs. Recently, the biomimetic cell membrane-camouflaged nanoparticles containing outstanding features have been widely used to detect and isolate rare cells from the peripheral blood samples. In this work, enythrocyte (RBC) and leukocyte (WBC) membranes are fused and coated onto magnet nanoparticles and then modified with anti-CD147 to isolate fNRBCs from the maternal peripheral blood with significant efficiency (∼90%) and purity (∼87%) in simulated spiked blood samples. Further, fNRBCs were isolated and identified from a series of maternal peripheral blood samples coming from pregnant women of 11-13 gestational weeks, and different chromosomal aneuploidies were diagnosed using fNRBCs isolated from maternal blood in early pregnancy. Our strategy may offer additional opportunity to overcome the limitations of current cell-based NIPD platforms.

Identification of Chemo-Resistant Residual Cell Population in Pediatric AML of Complete Remission By Single Cell RNA Sequencing

Although majority of pediatric acute myeloid leukemia (pAML) undergo complete remission (CR) after chemotherapy, more than 30% of which eventually relapse, leading to a dismal outcome. Chemo-resistant cells at CR, measured as minimal residual disease (MRD), are thought to be origin for relapse. Although more sensitive detection of MRD has been achieved through genetic mutations, the pathological and clinical significance for most of the detected mutations remain unknown, largely due to a lack of understanding for the biological context where the mutation resides. The idea of retention of leukemia stem cells (LSCs) as a mechanism of chemo-resistance has not been demonstrated post-therapy in patients. Here we applied single cell RNA sequencing (scRNA-seq) on 14 pAML who met the criteria for clinical CR (n=11) and partial remission (PR, n=3) to determine the cellular heterogeneity and cancerous feature of the residual cells that can survive chemotherapy at remission. These patients carried common genetic mutations such as AML1-ETO, CBFB-MYH11 and FLT3-ITD, which represents genetically diverse WHO subtypes. To maximize the power to detect tumor cells that can survive chemotherapy, we used 54 unsorted total bone marrow (BM) and/or peripheral blood (PB) nucleated cells collected at diagnosis and Day 26 (D26) of the first cycle of chemotherapy for 10X genomics' scRNA-seq. Cells from each pAML were compiled into one UMAP together with normal reference using unsupervised clustering to distinguish tumor clusters from normal. Clusters were defined to be tumor if patient's diagnosis contributed at least 80% of the cells, and were confirmed by the presence of somatic mutations and/or known AML mRNA expression signature associated with chromosome translocations. By projecting onto the closest normal hematopoietic cells based on transcription features, the tumor cells were classified as one of the 12 cell types (HSC/MPP, LMPP, GMP, MEP, E/B/M, CLP, classical/nonclassical Monocyte, cDC, pDC, and pre/inmature Neutrophil like cells). Consistently with findings in adult AML, cell populations were heterogeneous at diagnosis with 5-9 distinct clusters. Interestingly, majority (9/11) of the patients had 1-6 tumor clusters detected (mean 52 cells per cluster) at D26 post-chemotherapy. These D26 residual tumor cells possessed mutations originally detected by genomic sequencing and/or known AML signatures, and consistently clustered with tumor cells from diagnosis. These residual tumor cells accounted for average 1.4% of total BM cells at D26, while the morphological examination and flow cytometry analysis of MRD showed average 4.0% and 0.71% of tumor cells. To further evaluate the chemo-resistant potential of identified residual tumor cells, we focused on three distinct features known to be associated with chemo-resistance in mouse models, including LSC activity, active oxidative phosphorylation (OXPHOS) or leukemic-regenerating cell (LRC) state. Among the total 18 residual tumor clusters detected at D26, 33.3% (6/18) exhibited expression signatures associated with at least one chemo-resistant features. The remaining clusters consisted more differentiated progenitor/monocyte-like cells. Specifically, three patients (1 PR and 2 CR) had the HSC/MPP or LMPP-like clusters possessing strong LSC and OXPHOS-associated signatures. The remaining one patient had cDC-like cluster expressing reported LRC signature. Importantly, all these four patients had either unfavorable cytogenetics or persistence of driver mutations detected by PCR. Taken together, these data showed that pediatric AMLs represented heterogeneous populations at both diagnosis and remission. Among the residual tumor clusters that survived chemotherapy, a small fraction (6/18) were HSC/MPP-like, LMPP-like and cDC-like cells with known chemo-resistant expression features. These findings provide the first in vivo characterization of cellular heterogeneity in chemo-treated pAML with complete remission. Further studies are needed to determine the molecular characteristics of these residual cells that may convey chemo-resistance and to determine whether the presence of these cells are associated with increased risk of relapse. Disclosures No relevant conflicts of interest to declare.