The journey of neutropoiesis: how complex landscapes in bone marrow guide continuous neutrophil lineage determination

Neutrophils are the most abundant white blood cell, and differentiate in homeostasis in the bone marrow from hematopoietic stem cells (HSCs) via multiple intermediate progenitor cells into mature cells that enter the circulation. Recent findings support a continuous model of differentiation in the bone marrow of heterogeneous HSCs and progenitor populations. Cell fate decisions both at the level of proliferation and differentiation are enforced through expression of lineage-determining transcription factors (LDTFs) and their interactions, that are influenced by both intrinsic (intracellular) as well as extrinsic (extracellular) mechanisms. Neutrophil homeostasis is subjected to positive feedback loops, stemming from the gut microbiome, as well as negative feedback loops resulting from the clearance of apoptotic neutrophils by mature macrophages. Finally, the cellular kinetics regarding the replenishing of the mature neutrophil pool is discussed in light of recent, contradictory data.

Diagnosis and therapeutic decision-making for the neutropenic patient

Determining the cause of a low neutrophil count in a pediatric or adult patient is essential for the hematologist's clinical decision-making. Fundamental to this diagnostic process is establishing the presence or lack of a mature neutrophil storage pool, as absence places the patient at higher risk for infection and the need for supportive care measures. Many diagnostic tests, eg, a peripheral blood smear and bone marrow biopsy, remain important tools, but greater understanding of the diversity of neutropenic disorders has added new emphasis on evaluating for immune disorders and genetic testing. In this article, a structure is provided to assess patients based on the mechanism of neutropenia and to prioritize testing based on patient age and hypothesized pathophysiology. Common medical quandaries including fever management, need for growth factor support, risk of malignant transformation, and curative options in congenital neutropenia are reviewed to guide medical decision-making in neutropenic patients.

An improved culture protocol for the differentiation and survival of human promyelocytic leukaemia PLB-985 cell-line into mature neutrophil-like granulocytes

Circulating blood neutrophils are short-lived, lack proliferation capacity and cannot be transfected in vitro to express exogenous genes or proteins. These properties have made the ex vivo genetic manipulation of neutrophils challenging and hindered biochemical and molecular studies investigating the function of specific genes and proteins. Improved methodology for differentiating cell lines into mature neutrophil-like phenotypes, with similar morphological and functional properties to blood neutrophils would, therefore, be an important tool to probe the molecular properties of mature cells. The PLB-985 cell line was cultured in RPMI-1640 medium supplemented foetal calf serum (FCS) and penicillin/streptomycin. For induction of differentiation into neutrophil-like cells, the medium was supplemented with sodium pyruvate, N, N-dimethylformamide (DMF) and all-trans retinoic acid (ATRA), FCS and penicillin/streptomycin. The cytokines G-CSF and GM-CSF were used to enhance differentiation, prolong viability and delay the progression of the differentiated cells into apoptosis. The modified culture protocol and conditions induced PLB-985 cells to differentiate into mature, neutrophil-like granulocytes that resembled the morphology of mature blood neutrophils as evident by acquisition of a multi-lobed nucleus and granulated cytoplasm. These modified culture conditions resulted in enhanced differentiation into neutrophil-like cells and the apoptosis of these differentiated cells was delayed by supplementation with cytokines. This experimental system should be useful for studies probing the function of specific genes and proteins in human neutrophils.

Identification and characterization of human CD34+ and CD34dim/- neutrophil committed progenitors

We report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitors within SSClowCD45dim CD34+ and CD34dim/- bone marrow cells, that we named neutrophil myeloblast (NMs). CD34+ and CD34dim/- NMs resulted as either CD45RA+ or CD45RA-, with CD34+ CD45RA- NMs found as selectively expanded in chronic-phase chronic myeloid leukemia patients. By scRNA-seq experiments, CD34+ and CD34dim/- NMs were found to consist of combinations of four cell clusters, characterized by different maturation stages and distributed along two differentiation routes. Cell clusters were identified by neutrophil-specific gene profiles, one of them associated to an interferon-stimulated gene (ISG) signature, hence supporting recently identified expansions of mature neutrophil subsets expressing ISGs in blood of diseased individuals. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

A study to evaluate the diagnostic value investigations in early diagnosis of septicemia in newborn

Background: The early diagnosis of neonatal septicemia still poses great difficulties. Early clinical symptomatology of neonatal septicemia is mimicked by a lot of other disorders affecting the newborn. To study the diagnostic value of the combination of CRP, absolute neutrophil count, band form count to the total neutrophil ratio in early diagnosis of neonatal septicemia.Methods: A total 75 babies who got admitted to Mahavir institute of medical sciences between October 2018 to September 2019 (12) months were included in the study medical college hospital in a newborn with age less than 3 days and with well-defined maternal risk factors or clinical evidence of sepsis are included in the study. In all neonates, the blood sample was collected from a peripheral vein with all aseptic precautions, before administration of any antibiotic therapy.Results: A total 20 preterm babies (62.5%) were affected by septicemia.12 full-term babies (37.5%) were affected by septicemia. The sensitivity of a lab test is defined as the proportion of infants with proven sepsis in whom the result is abnormal. Specificity is the proportion of healthy infants in whom the result is normal.Conclusions. When both CRP and band form to mature neutrophil counts were positive the sensitivity and the negative predictive value were high compared to other combinations of two.

A neutrophil activation signature predicts critical illness and mortality in COVID-19

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of more than 3300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, hepatocyte growth factor, interleukin-8, and granulocyte colony-stimulating factor, which were the strongest predictors of critical illness. Evidence of neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, these data suggest a central role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular markers that distinguish patients at risk of future clinical decompensation.

A neutrophil activation signature predicts critical illness and mortality in COVID-19

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of over 3,300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, HGF, IL-8, and G-CSF, as the strongest predictors of critical illness. Neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, we define an essential role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular neutrophil markers that distinguish patients at risk of future clinical decompensation.

Immunogenomic identification and characterization of granulocytic myeloid-derived suppressor cells in multiple myeloma

Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14−CD15+CD33+HLADR− cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC–related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.

Haematological Parameters in Early Diagnosis of Clinically Suspected Neonatal Septicemia

Neonatal septicemia is a life-threatening yet treatable condition. It is one of the major health problems throughout the world. Blood culture is the gold standard for diagnosis of neonatal septicemia and should be performed in all cases of suspected septicemia prior to starting antibiotics. But in the present study, the use of the haematological parameters was evaluated to determine the early diagnosis of neonatal septicemia. It was carried out in the neonatal unit of Mandalay Children’s Hospital from September 2012 to August 2013. Out of 68 cases of clinically suspected neonatal septicemia, bacterial pathogens were isolated from 33 cases. Early onset neonatal septicemia was more common than late onset. Among 33 cases of culture-proven neonatal septicemia, septicemia is not likely in 1 case (3%) while borderline for septicemia and septicemia in 6 cases (18.2%) is very likely in 26 cases (78.8%) according to haematological scoring system (HSS). HSS assigns a score of one for each of seven haematological parameters. Out of the seven haematological parameters, immature to total neutrophil ratio (I:T) and immature to mature neutrophil ratio (I:M) were the most reliable test for the neonatal septicemia. Thrombocytopenia had low sensitivity. The haematolgical parameters are simple, quick, cost effective and readily available. The sensitivity, specificity and the predictive values of haematological parameters could be enhanced by combination of these parameters rather than the use of single parameter.

AutomatedPlasmodiumdetection by the Sysmex XN hematology analyzer

BackgroundMalaria is a potentially severe disease affecting nearly 200 million people per year. Early detection of the parasite even in unsuspected patients remains the challenging aim for effective patient care. Automated complete blood counts that are usually performed for any febrile patient might represent a tool to ascertain malaria infection.AimsTo evaluate the ability of the new generation of the Sysmex hematology analyzer (XN-series) to detect malaria.MethodsWe retrospectively studied 100 blood samples performed with the recent Sysmex XN analyzer that were positive forPlasmodiumand explored its ability to detect the parasite. 100 samples from patients uninfected by malaria were used as control group.ResultsSpecific abnormalities such as additional events in the mature neutrophil/eosinophil area of the white blood cells differential (WDF) scattergram were noted for 1.1% ofPlasmodium falciparumsamples and 56.2% of otherPlasmodiumspecies samples. Mature parasite stages (schizonts or gametocytes) were observed on blood smears among those samples. WDF scattergrams were able to detect 80.0% (12/15) ofPlasmodiummature stages. Furthermore, the differential in white blood counts between WDF and white cell nucleated (WNR) channels was a predictive signal ofPlasmodiummature stages in 73.3% (11/15) of samples and may be explained by a differential destruction of particles with the analyzer reagent.ConclusionAssociated to thrombocytopaenia, a Sysmex XNPlasmodiumpattern may represent a useful warning forPlasmodiumdetection in unsuspected patients, particularly when mature parasite stages are present.