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Background In acute myeloid leukemia controversy exists about the role of immunophenotyping of the blasts at diagnosis as a potential prognostic factor. Methods We retrospectively analyzed immunophenotypic marker expression on blasts in relation to genetic aberrancies and survival data of 684 patients. All patients were included in different studies from the HOVON/SAKK Consortium. Results Markers CD2, CD7, CD11b, CD19, CD22 and CD56 all appeared to be associated with one or more established prognostic genetic aberrancies. In the overall population, differences in univariate survival analyses were observed for CD2, CD11b and CD22. After correcting these survival differences for currently used risk profile data, only CD11b expression remained of prognostic value for poor overall survival (OS) and event free survival (EFS). CD11b expression turned out to be an independent factor for poor OS and EFS in the subgroup of patients who lacked cytogenetic and molecular aberrancies. Conclusions In this study we demonstrate that associations between immunophenotypic markers and genetic aberrancies interfere with the prognostic properties of immunophenotypic markers. Such may account for most of the previously reported prognostic impact of these markers. Only CD11b expression remained as an independent prognostic marker. This article is protected by copyright. All rights reserved.

Aims Male neonates appear to be more susceptible to infection than females. The immunological reasons for this are poorly understood. Our aim was to compare the immune response of healthy term male and female neonates using flow cytometry and cytokine analysis. Methodology Whole blood was collected with routine phlebotomy in healthy, term infants on the postnatal ward within 48 hours of delivery. Samples were processed for flow cytometry and cytokine analysis. Whole blood was treated with endotoxin (LPS; 10 ng/mL) for 1 hour at 37°C before subsequent analysis. Granulocytes and monocytes were identified by flow cytometry based on light scattering properties and cell surface markers. Multiplex ELISA was undertaken using a multisport 96-well plate customised for the experiment. Results In total 26 neonates were analysed (13 of each sex) with 20 having flow cytometry analysis and 6 undergoing ELISA. Female monocytes had higher CD11B expression than males following endotoxin stimulation. GM-CSF, IL-10, IL-1RA and IL-8 increased significantly following endotoxin stimulation in females but not males (p Conclusion Male and female neonates mount different immune responses following septic challenge. Results suggest that females mount a more robust immune response and may be better able to modulate the immune response resulting from infectious stimuli.

SummaryA growing body of evidence suggests a pivotal role of inflammatory processes in AF in a bidirectional manner. Infiltrating leukocytes seem to promote both structural and electrical remodelling processes in patients with AF. Monocyte-platelets-aggregates (MPAs) are sensitive markers of both platelets and monocyte activation. So far it is not clear whether the content of MPAs is affected by AF. The present study examined the content of MPAs and the activation of monocytes in elderly patients with an aortic stenosis in dependence of AF. These patients are known to have a high prevalence of AF. Flow-cytometric quantification analysis demonstrated that patients with AF have an increased content of MPAs (207 ± 13 cells/μl vs 307 ± 21 cells/μl, pSupplementary Material to this article is available online at www.thrombosis-online.com.

Introduction: LAD-I is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from ITGB2 gene mutations encoding for the β2-integrin component, CD18. Severe LAD-I (i.e., CD18 expression on Methods: Pediatric patients ≥ 3 months old with severe LAD-I (demonstrated by CD18 expression on Results: Two patients (ages 9 and 3) were treated in Phase 1. Both have a history of recurrent severe infections, documented ITGB2 mutations, and baseline CD18, CD11a, and CD11b expression < 1%. Mobilization and apheresis procedures were performed successfully and busulfan was administered at the target AUC. Investigational product comprised of 4.2x106 CD34+ cells/kg with VCN of 3.8 copies/cell (liquid culture) for Patient 1 and 2.8x106 CD34+ cells/kg with VCN of 2.5 for Patient 2 and were infused without complications. No serious treatment-emergent adverse events were reported. Neutrophil engraftment was observed in < 3 weeks in both patients. For Patient 1, PB PMN CD18 expression 6 months post-treatment was 47% (sustained from 45% at 3-months, vs. < 1% at baseline), with PB VCN of 1.3. Skin lesions present at baseline were resolving with no new lesions. Replication competent lentiviral (RCL) testing at 3 months and 6 months post-infusion were negative. Safety and efficacy data 12-months post-treatment for Patient 1 and 6-months post-treatment for Patient 2 will be available at the time of presentation. Conclusion: Initial results from Phase 1 demonstrate preliminary safety and efficacy of RP-L201 for reversal of severe LAD-I. Enrollment of patients in the Phase 2 study is underway. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Almarza:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Sevilla:Novartis: Other: Advisory Board; Sobi: Other: Advisory Board; Rocket Pharma: Consultancy; Amgen: Other: Advisory Board. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company.

An inflammatory systemic reaction is common after Transcatheter Aortic Valve Implantation (TAVI). We recently reported about an involvement of Mon2-monocytes, the CD11b expression on monocytes and parameters of systemic inflammation before TAVI correlating with early mortality after TAVI. Here, we provide data of monocyte subpopulations, CD11b expression and parameters of a systemic inflammation in dependence of three-month mortality after TAVI. With this, we provide further insights into inflammatory mechanism after TAVI. The data were collected by flow-cytometric quantification analyses of peripheral blood in 120 consecutive patients who underwent TAVI (on day 1 and 7 after TAVI). Monocyte-subsets were identified by their CD14 and CD16 expression and monocyte-platelet-aggregates (MPA) by CD14/CD41 co-expression. The extent of monocyte activation was determined by quantification of CD11b-expression (activate epitope). Additionally, pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, C-reactive protein, procalcitonin were measured using the cytometric bead array method or standard laboratory tests. Additionally, we report procedural outcomes in dependence of three-month mortality. Furthermore, correlations of CD11b-expression on monocytes with parameters of platelet activation or further inflammatory parameters are presented. For further interpretation of the presented data, please see the research article “Mon2-Monocytes and Increased CD-11b Expression Before Transcatheter Aortic Valve Implantation are Associated with Earlier Death” by Pfluecke et al. [1]

This is the pre-peer reviewed version of the following article: Hardersen, R., Enebakk, T., Christiansen, D., Bergseth, G., Brekke, L.-O., Mollnes, T.E., ... Hovland, A.W. (2018). Granulocyte and monocyte CD11b expression during plasma separation is dependent on complement factor 5 (C5) ? an ex vivo study with blood from a C5-deficient individual. Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), 126(4), 342-352, which has been published in final form at https://doi.org/10.1111/apm.12821. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. The aim of the study was to investigate the role of complement factor 5 (C5) in reactions elicited by plasma separation using blood from a C5‐deficient (C5D) individual, comparing it to C5‐deficient blood reconstituted with C5 (C5DR) and blood from healthy donors. Blood was circulated through an ex vivo plasma separation model. Leukocyte CD11b expression and leukocyte–platelet conjugates were measured by flow cytometry during a 30‐min period. Other markers were assessed during a 240‐min period. Granulocyte and monocyte CD11b expression did not increase in C5D blood during plasma separation. In C5DR samples granulocytes CD11b expression, measured by mean fluorescence intensity (MFI), increased from 10481 ± 6022 (SD) to 62703 ± 4936, and monocytes CD11b expression changed from 13837 ± 7047 to 40063 ± 713. Granulocyte–platelet conjugates showed a 2.5‐fold increase in the C5DR sample compared to the C5D sample. Monocyte–platelet conjugates increased independently of C5. In the C5D samples, platelet count decreased from 210 × 109/L (201–219) (median and range) to 51 × 109/L (50–51), and C3bc increased from 14 CAU/mL (21–7) to 198 CAU/mL (127–269), whereas TCC formation was blocked during plasma separation. In conclusion, up‐regulation of granulocyte and monocyte CD11b during plasma separation was C5‐dependent. The results also indicate C5 dependency in granulocyte–platelet conjugates formation.

Aim: Sepsis-induced disseminated intravascular coagulopathy is associated with a high mortality rate. The function and deformability of polymorphonuclear leukocytes change in patients with sepsis. The goal of this study was to characterize the changes in polymorphonuclear leukocyte deformability in patients with sepsis-induced disseminated intravascular coagulopathy and to evaluate the relationship between the severity of disseminated intravascular coagulopathy and the deformability of polymorphonuclear leukocytes. Methods: Thirty-five patients with sepsis-induced disseminated intravascular coagulopathy at our department were enrolled in this study. These patients were diagnosed with severe sepsis and an acute disseminated intravascular coagulopathy score ≥ 4. Blood samples were obtained from these patients on days 1, 3, and 7. Polymorphonuclear leukocyte deformability was measured with a microchannel flow analyzer, and polymorphonuclear leukocyte activity, represented as CD11b, was measured by flow cytometry. In contrast, 14 patients who fulfilled with sepsis criteria but without complicated disseminated intravascular coagulopathy were also entered in this study. Results: In patients with sepsis-induced disseminated intravascular coagulopathy, there was a significant correlation between their Japanese Association for Acute Medicine disseminated intravascular coagulopathy score and polymorphonuclear leukocyte deformability, and CD11b expression. Polymorphonuclear leukocytes became more stiffened and CD11b expression was higher in patients with sepsis-induced disseminated intravascular coagulopathy compared to patients without the condition. Conclusion: Polymorphonuclear leukocyte deformability correlated with the severity of sepsis-induced disseminated intravascular coagulopathy and the response to treatment.

Among other phenotypic markers, CD11b expression has been considered as an unfavorable prognostic factor, both in terms of overall survival (OS), disease-free survival (DFS), and attainment and duration of complete remissions (CRs) in adult patients with acute myeloid leukemia (AML). Recently, some groups have restricted its prognostic impact to poor prognostic karyotypic risk groups. The aim of this study was to retrospectively analyze the prevalence of CD11b and of CD56 expression in blast cells of 158 AML patients [excluding those with t(15;17)] stratified according to their cytogenetic risk and to correlate these phenotypic characteristics with OS, DFS, and CR. CD11b was more frequently expressed in intermediate and unfavorable cytogenetic prognostic groups (38.9 and 35.5 %, respectively) than in the favorable group (9.5 %). No differences were observed in CD56 expression according to the cytogenetic risk groups. When OS, DFS, and CR were analyzed according to these two markers, no statistical differences were recorded in any cytogenetic risk group. In conclusion, although CD11b was more frequently expressed in blast cells of patients with intermediate and unfavorable cytogenetic risk groups, this feature did not translate into different clinical outcome. Similarly, CD56 positivity did not have any influence on the prognosis of these patients.

Summary Introduction Changes in leukocyte cell population data have been reported in various infectious diseases, but little is known in other inflammatory conditions such as the postprandial state. We investigated whether leukocyte cell population data change during postprandial leukocyte activation. Methods Healthy volunteers underwent a standardized oral fat loading test (OFLT). Flowcytometric quantitation of leukocyte activation markers CD11b, CD66b, CD35, and CD36, together with leukocyte cell population data from LH750 hematology analyzers were measured fasting and at 4 and 8 h postprandially. Results Twelve volunteers were included. Postprandial leukocyte activation was confirmed by increased expression of CD11b by monocytes (+11.7%) and neutrophils (+15.0%) and by increased expression of CD66b (+14.7%) and CD35 (+16.6%) by neutrophils at T = 4 h. The mean scatter from neutrophils, reflecting granularity, significantly decreased at T = 4 h (P

Background. The use of Di-2-ethylhexyl phthalate (DEHP) plasticised polyvinyl chloride (DEHPPPVC) in medical devices persists despite evidence suggesting that DEHP migration can be harmful. Researchers have shown that a simple surface sulfonation process can retard the migration of DEHP, which may reduce the associated inflammatory response. The present study is designed to investigate the effects of surface sulfonation on DEHP migration and blood contact activation using in vitro and rodent models. Methods. The study was carried out in two phases: phase 1, in which the migration rate of DEHP from DEHPPPVC and sulfonated DEHP plasticised PVC (SDEHPPPVC) was measured; phase 2 of the study, in which the materials were incorporated into a rat recirculation biomaterial test model and blood samples taken to assess CD11b expression on neutrophils, IL-6 and Factor XIIa. Results. The initial DEHP concentration washed from the surface after storage was 37.19 ± 1.17 mg/l in the PPVC group and 5.89 ± 0.81 mg/l in the SPPVC group (p Conclusions. The simple sulfonation process significantly retards the migration of DEHP and is associated with the moderation of contact activation processes.

Objective. Activated neutrophils have been implicated as effectors of tissue injury and end-organ damage in adult respiratory distress syndrome and multi-organ dysfunction syndrome. Recent research has shown that hypertonic saline may abrogate neutrophil activation and in turn attenuate end-organ damage, while lactated Ringer's solution causes neutrophil activation. There has been little research investigating the effects of iso-osmolar solutions such as 0.9% saline on neutrophil activation. The aim of this study was to determine the effect of an infusion of 0.9% saline on the activation of human neutrophils in healthy volunteers. Material and methods. A 2-l bolus infusion of 0.9% saline was administered to eight healthy volunteers over a 1-h period after an overnight fast. Venous blood samples were taken on three occasions: pre-infusion (time 0), on completion of the infusion (time 1) and 1 h later (time 2). Neutrophil activation was quantified by flow cytometric analysis of CD11b expression. Submaximal ...

Our objective was to assess whether using a methanol wash to reduce the level of plasticizer present on the surface of medical-grade polyvinyl chloride (PVC) has a moderating effect on the expression of CD11b (mac-1) on neutrophils in rats undergoing recirculation. The study was carried out on 3 groups of 10 adult male Sprague-Dawley rats weighing between 350 and 450 g. In the 2 test groups, the animals were exposed to 48 cm2 of di-(2-ethyl-hexyl)-phthalate (DEHP)-plasticized PVC in a parallel plate recirculating test cell through which blood was recirculated at 1.5 ml/min. In the first test group, the PVC was untreated; in the second test group, the PVC was washed in methanol to reduce the level of plasticizer on the surface. The test cell was connected to the right femoral circulation, and recirculation was established for a period of 60 min. Blood samples were taken at 0, 30, and 60 min for assessment of CD11b expression on neutrophils using flow cytometric analysis. In a third group of 10 control experiments, rats underwent the entire surgical procedure, but without recirculation through the test cell. There was statistically significant (p < 0.001) lower Cdllb expression on neutrophils in the blood of rats perfused through the cell containing methanol-washed PVC after 30 min and at 60 min. CD11b expression was significantly (p < 0.001) lower in the control group than in both test groups at both the 30 and 60 min time points and at the 60 min time point on comparison with the group where blood was perfused through methanol-washed PVC. These results demonstrate that the biomaterial-contact-mediated upregulation of CD11b may be significantly reduced by employing a methanol-washing technique on the plasticized PVC. Although this technique does not entirely eliminate the expression of CDllb on neutrophils, the difference is significant and suggests the role of the plasticizer in the development of this inappropriate inflammatory response.

To the Editor Although the unequivocal diagnosis of acute promyelocytic leukemia (APL) is established by the finding of t(15;17) and/or the PML / RARA fusion gene, cytogenetic and molecular techniques are time-consuming. Instead, flow cytometry (FC) allows a prompt diagnosis of this form of leukemia and the rapid institution of the specific therapy. Several studies have tried to identify the panel of markers that better characterize this entity. In a series of 149 patients with APL, Dong et al1 found as constant the triple negativity for HLA-DR, CD11b, and CD11c. Similar results were obtained in a shorter study that analyzed 16 cases of APL,2 in which 100% of the patients were negative for CD34, CD14, and CD11b (CD11c was not studied). We hereby share a different experience in a similar study. The expression of CD34, CD11b, and DR, among other markers, was analyzed by FC in a retrospective series (years 2008–2013) of 18 patients (7 …

The objectives of this study were to (1) determine chemotaxis (CX) response by neutrophils (PMNs) isolated from patients with the localized form of pre-pubertal periodontitis (L-PP) and compare these responses with those of age-matched and adult controls, (2) determine the level and up-regulation of CDllb expression upon stimulation with fMLP by peripheral blood PMNs (PB-PMNs) isolated from patients with L-PP and compare these levels with those of age-matched and adult controls, and (3) determine whether there is a correlation between CX and CDllb expression (up-regulation) by PB-PMNs. PB-PMNs from a total of seven patients with L-PP, seven age-matched pediatric controls, and 11 adult controls (four adults for both CDllb and CX assays, and seven adults for CX assays only) were analyzed for CX and CDllb expression. Direct immunofluorescence staining of CDllb was performed with FITC-conjugated monoclonal antibody. Flow cytometry was used to analyze stained cells for their fluorescence intensity. Chemotaxis activity in response to 10-8 mol/L fMLP was examined in micro-well chemotaxis chambers. The results indicated that CX of PMNs from both L-PP patients and pediatric control subjects was significantly decreased, compared with that of normal adult control subjects (p0.7). CDllb expression did not significantly differ among L-PP patients, normal pediatric controls, and adult controls. Analysis of the data indicates that there was no statistically significant correlation between CX and CDllb expression by PMNs isolated from L-PP and pediatric control subjects.

To observe the effect of ketamine on polymorphonuclear leucocytes (PMN) adhesion and respiratory burst activity in endotoxemia rats.30 rats were randomly allocated to five groups: rats challenged with intraperitoneal injection of saline (saline group); challenged with intraperitoneal injection of LPS 10 mg/kg (LPS group); challenged with intraperitoneal injection of LPS 10 mg/kg and treated by intraperitoneal injection of ketamine 5, 25, 50 mg/kg at 0, 1, 2, 3, 4, 5 h after the injection of LPS, respectively (three ketamine treatment groups).PMN respiratory burst and CD11b expression were measured with flow cytometry at the end of 1 h, 4 h, and 6 h.LPS challenge significantly increased PMN respiratory burst activity and CD11b expression when compared with the saline group (p0.01). There was a significant decrease in LPS-induced PMN respiratory burst activity and CD11b expression in three ketamine treatment groups when compared with LPS group (p0.01).Ketamine significantly inhibits PMN CD11b expression and respiratory burst activity in endotoxemic rats.

Background: Dysfunctional inflammatory responses have been implicated in several neonatal inflammatory disorders following infection and hypoxia. Objectives: We aimed to study the effects of in vitro hypoxia and heat shock (HS) on normal adult and newborn neutrophil migration (CD11b) and persistence (apoptosis) following lipopolysaccharide (LPS) stimulation. Methods: The mechanism for altered LPS responses was assessed at the level of the LPS signalling receptors, Toll-like receptor-4 (TLR-4), TLR-2 and CD14 expression in normal neonates and adults. Results: In adults, although hypoxia delayed neutrophil apoptosis, LPS enhanced this response. In contrast, HS (42°C) increased adult apoptotic rates and abrogated the LPS responses. Both hypoxia and HS prevented the LPS-induced increase in adult CD11b although it was unaltered in neonates. Adult TLR-4 neutrophil expression was increased by LPS and hypoxia, and decreased in HS, possibly explaining their variable LPS responsiveness. In contrast, neonatal neutrophils were LPS hyporesponsive which may be mediated by failure of TLR-4 upregulation with LPS. Conclusions: Neonates do not have increased LPS responsiveness in hypoxia or heat shock in vitro, which may prevent hyperinflammation and thereby minimise tissue damage in inflammation or infection.

An up-regulation of the surface marker CD11b has been demonstrated during polymorphonuclear (PMN) cell activation. CD11b over-expression is often associated with inflammation and is considered as an early marker of infection. However, the absence of standardized assay and the variability of preanalytical settings leading to PMN artifactual activation have compromised the interest of this marker. In the present study a standardized quantitative flow cytometry assay directly performed in whole blood has been used to determine CD11b expression on PMN cells. The results indicate that quantitative flow cytometry can provide consistent CD11b density values between laboratories provided that a calibration system is used including specific calibrators, reagents and protocols. This method allowed us to evidence an up-regulation of CD11b expression for infected patients. This quantitation is a standardized and potentially useful method in clinical situations implying quantitative CD11b expression variations.

Gamma interferon (IFN-gamma) has multiple immunomodulating effects and has been postulated as a possible immunopotentiating agent for the prevention or treatment of neonatal infections. This report describes the effect of rat recombinant IFN-gamma on the oxidative burst activity and CD11b expression of neonatal and adult rat polymorphonuclear leukocytes (PMNL). Oxidative burst activity was assessed by chemiluminescence and dihydrorhodamine flow cytometry. Neonatal PMNL exhibited significantly less oxidative burst activity than did adult PMNL. IFN-gamma mildly enhanced the chemiluminescence response of PMNL from both the rat pups and adults, but this effect was not statistically significant when analyzed by a multivariate model of repeated-measures analysis of variance for both chemiluminescence and dihydrorhodamine flow cytometry. CD11b expression was also not significantly enhanced by IFN-gamma.

Mean fluorescence intensity (MFI) of beta2 integrin (CD11a and CD11b) expression on peripheral blood (PB) CD34-positive (CD34 + ) cells in steady state correlates inversely with the total amount of harvested CD34 + cells

This study investigated the cell surface expression of CD11b (Mo 1, Mac-1, CR 3) by neutrophils (PMNs) from human crevicular fluid (CF-PMNs). CD11b expression on CF-PMNs was compared, using flow cytometry, to that on peripheral blood PMNs (PB-PMNs) isolated simultaneously from the same subjects. CD11b expression by CF-PMNs was also compared to that on PB-PMNs stimulated with formylmethionylleucylphenylalanine (fMLP) or crevicular fluid supernatant (cell-free portion of CF diluted with buffer) (CFS). Crevicular PMNs consistently expressed more CD11b than unstimulated PB-PMNs (p less than 0.001). The level of CD11b expressed on these CF-PMNs was significantly greater than that expressed by PB-PMNs stimulated with 10(-8) M fMLP (p less than 0.05). The level of CD11b surface expression on CF-PMNs was not statistically different from that expressed on PB-PMNs stimulated with CFS or 10(-6) M fMLP. However, CFS significantly stimulated upregulation of surface CD11b expression on PB-PMNs relative to that achieved with 10(-8) M fMLP (0.01 less than p less than or equal to 0.025). Thus it is concluded that CF-PMNs express a high level of CD11b and CFS can upregulate CD11b expression on PB-PMNs to a level that is at least as great as that achieved with 10(-8) M fMLP.