Fragile neutrophils in surgical patients: A phenomenon associated with critical illness.

Leukocyte viability (determined by e.g. propidium iodide [PI] staining) is automatically measured by hematology analyzers to check for delayed bench time. Incidental findings in fresh blood samples revealed the existence of leukocytes with decreased viability in critically ill surgical patients. Not much is known about these cells and their functional and/or clinical implications. Therefore, we investigated the incidence of decreased leukocyte viability, the implications for leukocyte functioning and its relation with clinical outcomes. An automated alarm was set in a routine hematology analyzer (Cell-Dyn Sapphire) for the presence of non-viable leukocytes characterized by increased fluorescence in the PI-channel (FL3:630±30nm). Patients with non-viable leukocytes were prospectively included and blood samples were drawn to investigate leukocyte viability in detail and to investigate leukocyte functioning (phagocytosis and responsiveness to a bacterial stimulus). Then, a retrospective analysis was conducted to investigate the incidence of fragile neutrophils in the circulation and clinical outcomes of surgical patients with fragile neutrophils hospitalized between 2013-2017. A high FL3 signal was either caused by 1) neutrophil autofluorescence which was considered false positive, or by 2) actual non-viable PI-positive neutrophils in the blood sample. These two causes could be distinguished using automatically generated data from the hematology analyzer. The non-viable (PI-positive) neutrophils proved to be viable (PI-negative) in non-lysed blood samples, and were therefore referred to as 'fragile neutrophils'. Overall leukocyte functioning was not impaired in patients with fragile neutrophils. Of the 11 872 retrospectively included surgical patients, 75 (0.63%) were identified to have fragile neutrophils during hospitalization. Of all patients with fragile neutrophils, 75.7% developed an infection, 70.3% were admitted to the ICU and 31.3% died during hospitalization. In conclusion, fragile neutrophils occur in the circulation of critically ill surgical patients. These cells can be automatically detected during routine blood analyses and are an indicator of critical illness.

On the origin of low-density neutrophils.

Here we elaborate on the origin of low(er)-density neutrophils (LDNs) to better understand the variation found in literature. Supplemented with original data, we test the hypothesis that buoyant density of neutrophils is characterized by a spectrum that as a whole shifts to a lower density after activation. Both the 20% highest density (HDNs) and 20% lowest density (LDNs) neutrophils from healthy donors were isolated by Percoll of different densities. Using this method we found that LDNs were significantly better in T-cell suppression and bacterial containment than their 20% highest density counterparts. We found no statistically relevant differences in neutrophil survival or bacterial phagocytosis. Stimulation of healthy donor neutrophils with N-formyl-methionyl-leucyl-phenylalanine induced LDNs co-segregating with peripheral blood mononuclear cells after Ficoll separation. These in vitro induced LDNs showed increased activation markers compared to HDNs and were comparable to the activation markers found on the LDN fraction seen in patients with chronic inflammatory conditions such as present in cancer patients. This all fits with the hypothesis that the density of neutrophils is distributed in a spectrum partially coupled to maturation. Additionally a shift in this spectrum can be induced by in vitro stimulation or by activation in disease.

The effect of early broad-spectrum versus delayed narrow-spectrum antibiotic therapy on the primary cure rate of acute infection after osteosynthesis.

PURPOSE: Infection near metal implants is a problem that presents challenging treatment dilemmas for physicians. The aim of this study was to analyse the efficacy of two treatment protocols for acute fracture-related infections. METHODS: Seventy-one patients in two level-1 trauma centres in the Netherlands were retrospectively included in this study. These trauma centres had different standardised protocols for acute infection after osteosynthesis: 39 patients were selected from protocol A and 32 from protocol B. Both protocols involve immediate surgical debridement and soft tissue coverage, but differ in antibiotic approach: (A) immediate empirical combination antibiotic therapy with rifampicin, or (B) postponed (1-5 days) targeted antibiotic therapy. The primary outcome of these protocols was success, defined as a fracture healing in the absence of infection. The secondary outcome was antibiotic resistance patterns. Logistic regression was conducted on patients and treatment-related factors in association with primary success. RESULTS: Primary success was achieved in 72% of protocol A patients, in 47% of those in protocol B (P = 0.033), and with prolongation of treatment success was achieved in 90% and 78% of patients, respectively. Protocol A exhibited a better primary success rate (adjusted OR 3.45, CI 1.13-10.52) when adjusted for age and soft tissue injury. There was no significant difference in antibiotic resistance between the two protocols. CONCLUSION: Both protocols yielded high overall success rates. Immediate empirical antibiotics can be used safely without additional bacterial resistance and may contribute to increased success rates.

Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes.

INTRODUCTION: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. METHODS: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. RESULTS: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×-2.2×) p = .058, CD11b: 2.13× (1.6×-2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed. CONCLUSION: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15-20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Intracellular Penetration and Effects of Antibiotics on Staphylococcus aureus Inside Human Neutrophils: A Comprehensive Review.

Neutrophils are important assets in defense against invading bacteria like staphylococci. However, (dysfunctioning) neutrophils can also serve as reservoir for pathogens that are able to survive inside the cellular environment. Staphylococcus aureus is a notorious facultative intracellular pathogen. Most vulnerable for neutrophil dysfunction and intracellular infection are immune-deficient patients or, as has recently been described, severely injured patients. These dysfunctional neutrophils can become hide-out spots or "Trojan horses" for S. aureus. This location offers protection to bacteria from most antibiotics and allows transportation of bacteria throughout the body inside moving neutrophils. When neutrophils die, these bacteria are released at different locations. In this review, we therefore focus on the capacity of several groups of antibiotics to enter human neutrophils, kill intracellular S. aureus and affect neutrophil function. We provide an overview of intracellular capacity of available antibiotics to aid in clinical decision making. In conclusion, quinolones, rifamycins and sulfamethoxazole-trimethoprim seem very effective against intracellular S. aureus in human neutrophils. Oxazolidinones, macrolides and lincosamides also exert intracellular antibiotic activity. Despite that the reviewed data are predominantly of in vitro origin, these findings should be taken into account when intracellular infection is suspected, as can be the case in severely injured patients.

A Rise in Neutrophil Cell Size Precedes Organ Dysfunction After Trauma.

INTRODUCTION: Organ dysfunction remains a major cause of morbidity after trauma. The development of organ dysfunction is determined by the inflammatory response, in which neutrophils are important effector cells. A femoral fracture particularly predisposes for the development of organ dysfunction. This study investigated the chronologic relation between neutrophil characteristics and organ dysfunction in trauma patients with a femoral fracture. METHODS: Patients with a femoral fracture presenting at the University Medical Center Utrecht between 2007 and 2013 were included. Data of neutrophil characteristics from standard hematological analyzers were recorded on a daily basis until the 28th day of hospital stay or until discharge. Generalized Estimating Equations were used to compare outcome groups. RESULTS: In total 157 patients were analyzed, of whom 81 had polytrauma and 76 monotrauma. Overall mortality within 90 days was 6.4% (n = 10). Eleven patients (7.0%) developed organ dysfunction. In patients who developed organ dysfunction a significant increase in neutrophil count (P = 0.024), a significant increase in neutrophil cell size (P = 0.026), a significant increase in neutrophil complexity (P < 0.004), and a significant decrease in neutrophil lobularity (P < 0.001) were seen after trauma. The rise in neutrophil cell size preceded the clinical manifestation of organ dysfunction in every patient. CONCLUSION: Patients who develop organ dysfunction postinjury show changes in neutrophil characteristics before organ dysfunction becomes clinically evident. These findings regarding post-traumatic organ dysfunction may contribute to the development of new prognostic tools for immune-mediated complications in trauma patients. LEVEL OF EVIDENCE: Level II, etiologic study.

Neutrophil phenotypes in health and disease.

Neutrophils are one of the most important effector cells of the innate immune response (1). They are traditionally seen as a homogenous population of short-lived cells mainly involved in the defence against extracellular microorganisms by phagocytosis and intracellular killing (1,2). The cells contain a large armamentarium that aids in this function and ranges from the production of reactive oxygen species by a membrane-bound NADPH oxidase to cytotoxic proteins and peptides residing in the different granules present in the cytoplasm (3). Recently, the view of neutrophils belonging to a homogenous population of cells has been challenged, and several neutrophil phenotypes have been described that exhibit specialized functions, such as involvement in tissue repair, tumour killing and immune regulation (4). It is not clear whether these cells belong to separate parallel lineages originating from the bone marrow or that neutrophils become instructed in the distant tissues, thus changing their phenotypes. In addition, functional heterogeneity in a phenotypically homogenous population of neutrophils adds to the complexity of neutrophil phenotypes(5). This article will review the current literature describing the heterogeneity within the neutrophil compartment with respect to both phenotype and function in health and disease.

Neutrophil Functional Heterogeneity: Identification of Competitive Phagocytosis.

INTRODUCTION: Phagocytosis by neutrophils is a key process in the innate immune response against invading microorganisms. Despite reported heterogeneity in other neutrophils functions, little is known regarding differences in phagocytosis by individual cells. Therefore, we tested the hypothesis that heterogeneity is present in the neutrophil compartment in its potency to phagocytize bacteria. METHODS: Phagocytosis assays were performed in suspension with isolated neutrophils and Staphylococcus aureus expressing different fluorescent proteins at MOIs between 1 and 10. Repetitive addition of bacteria with different fluorescent proteins and MOIs was used to compare the phagocytic capacity of S. aureus-green fluorescent protein (GFP)-positive and negative neutrophils and exclude randomness. RESULTS: The percentage and mean fluorescence intensity (MFI) of S. aureus-GFP-positive neutrophils increased with higher MOIs. The increase in MFI was due to phagocytosis of multiple bacteria per neutrophil as was confirmed by confocal imaging. Sequential phagocytosis of GFP- and mCherry-expressing S. aureus showed a non-random process, as S. aureus-GFP-positive neutrophils preferentially phagocytized S. aureus-mCherry. CONCLUSION: All neutrophils were able to phagocytize S. aureus, but some were much more potent than others. Therefore, at physiologically relevant MOIs these potent phagocytizing neutrophils will outcompete the uptake of bacteria by less competent cells in a process we propose to name "competitive phagocytosis."

Effect of a standardized treatment regime for infection after osteosynthesis.

BACKGROUND: Infection after osteosynthesis is an important complication with significant morbidity and even mortality. These infections are often caused by biofilm-producing bacteria. Treatment algorithms dictate an aggressive approach with surgical debridement and antibiotic treatment. The aim of this study is to analyze the effect of such an aggressive standardized treatment regime with implant retention for acute, existing <3 weeks, infection after osteosynthesis. METHODS: We conducted a retrospective 2-year cohort in a single, level 1 trauma center on infection occurring within 12 months following any osteosynthesis surgery. The standardized treatment regime consisted of implant retention, thorough surgical debridement, and immediate antibiotic combination therapy with rifampicin. The primary outcome was success. Success was defined as consolidation of the fracture and resolved symptoms of infection. Culture and susceptibility testing were performed to identify bacteria and resistance patterns. Univariate analysis was conducted on patient-related factors in association with primary success and antibiotic resistance. RESULTS: Forty-nine patients were included for analysis. The primary success rate was 63% and overall success rate 88%. Factors negatively associated with primary success were the following: Gustilo classification (P = 0.023), higher number of debridements needed (P = 0.015), inability of primary closure (P = 0.017), and subsequent application of vacuum therapy (P = 0.030). Adherence to the treatment regime was positively related to primary success (P = 0.034). CONCLUSIONS: The described treatment protocol results in high success rates, comparable with success rates achieved in staged exchange in prosthetic joint infection treatment.