Identification of neutrophil phenotype categories in geriatric hip fracture patients aids in personalized medicine.

Objectives: The number of geriatric hip fracture patients is high and expected to rise in the coming years, and many are frail and at risk for adverse outcomes. Early identification of high-risk patients is crucial to balance treatment and optimize outcome, but remains challenging. Previous research in patients with multitrauma suggested that neutrophil phenotype analysis could aid in early identification of high-risk patients. This pilot study investigated the feasibility and clinical value of neutrophil phenotype analysis in geriatric patients with a hip fracture. Methods: A prospective study was conducted in a regional teaching hospital in the Netherlands. At the emergency department, blood samples were collected from geriatric patients with a hip fracture and analyzed using automated flow cytometry. Flow cytometry data were processed using an automated clustering algorithm. Neutrophil activation data were compared with a healthy control cohort. Neutrophil phenotype categories were assessed based on two-dimensional visual assessment of CD16/CD62L expression. Results: Blood samples from 45 geriatric patients with a hip fracture were included. Neutrophils showed an increased activation profile and decreased responsiveness to formyl peptides when compared to healthy controls. The neutrophil phenotype of all patients was categorized. The incidence of severe adverse outcome was significantly different between the different categories (P = 0.0331). Moreover, patients with neutrophil phenotype category 0 developed no severe adverse outcomes. Conclusions: Using point-of-care fully automated flow cytometry to analyze the neutrophil compartment in geriatric hip fracture patients is feasible and holds clinical value in determining patients at risk for adverse outcome. This study is a first step toward immuno-based precision medicine for identifying geriatric hip fracture patients that are deemed fit for surgery.

Helicopter-Based Search and Rescue Operations in the Dutch Caribbean: A Retrospective Analysis.

INTRODUCTION: Search and rescue (SAR) operations in the Dutch Caribbean offer basic and advanced prehospital care and transport for definitive care. Helicopter-based SAR in this geographic area has not been previously studied. Data from the Dutch Caribbean Coast Guard were analyzed with the aim of describing the current operational setting and optimizing SAR operations in the future. METHODS: Data were collected retrospectively from March 2018 through April 2021. Epidemiologic data, patient demographics, details of flight operations, medical interventions, and outcomes were collected and analyzed for this period. RESULTS: A total of 91 individuals were assisted through SAR, of whom 40 (44%) had a medical emergency. Most incidents occurred during high-tourism seasons. A yearly increase in helicopter tasking was observed. Boating was the most common activity (25%) requiring SAR. Injuries to the extremities were the most common injury (27%). The median time to reach the scene of SAR was 46 (interquartile range [IQR], 33-66) min. The most frequent reason for delay was the unavailability of a winch operator (15%). Of 16 fatalities, most (63%) were attributed to drowning. A total of 18 persons were transported to a hospital, with a median travel time of 63 (IQR, 47-79) min. CONCLUSIONS: The number of SAR missions in the Dutch Caribbean is increasing. The response times might be reduced by the inclusion of an on-call winch operator. A hospital helipad would likely decrease the time to definitive care. Stand-by physicians might improve the quality of medical care. Collection of data should be optimized in the future.

Shift of Neutrophils From Blood to Bone Marrow Upon Extensive Experimental Trauma Surgery.

Introduction: Extensive trauma surgery evokes an immediate cellular immune response including altered circulatory neutrophil numbers. The concurrent bone marrow (BM) response however is currently unclear. We hypothesize that these BM changes include (1) a relative reduction of the bone marrow neutrophil fraction and (2) increasing heterogeneity of the bone marrow neutrophil pool due to (3) the appearance of aged/returning neutrophils from circulation into the BM-compartment. Materials and Methods: Eight pigs were included in a standardized extensive trauma surgery model. Blood and bone marrow samples were collected at baseline and after 3 hours of ongoing trauma surgery. Leukocyte and subtype counts and cell surface receptor expression levels were studied by flow cytometry. Results: All animals survived the interventions. A significant drop in circulating neutrophil counts from 9.3 to 3.2x106 cells/ml (P=0.001) occurred after intervention, whereas circulatory neutrophil cell surface expression of CD11b increased. The concurrent bone marrow response included an increase of the BM neutrophil fraction from 63 ± 3 to 71 ± 3 percent (P<0.05). Simultaneously, the BM neutrophil pool became increasingly mature with a relative increase of a CXCR4high-neutrophil subtype that was virtually absent at baseline. Conclusion: The current study shows a shift in composition of the BM neutrophil pool during extensive trauma surgery that was associated with a relatively circulatory neutropenia. More specifically, under these conditions BM neutrophils were more mature than under homeostatic conditions and a CXCR4high-neutrophil subset became overrepresented possibly reflecting remigration of aged neutrophils to the BM. These findings may contribute to the development of novel interventions aimed to modify the trauma-induced immune response in the BM.

Kinetics of Neutrophil Subsets in Acute, Subacute, and Chronic Inflammation.

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.

The Systemic Immune Response in COVID-19 Is Associated with a Shift to Formyl-Peptide Unresponsive Eosinophils.

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (<100 cells/µL; p < 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6; p < 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.

Thrombotic Events in COVID-19 Are Associated With a Lower Use of Prophylactic Anticoagulation Before Hospitalization and Followed by Decreases in Platelet Reactivity.

Background: Coronavirus disease of 2019 (COVID-19) is associated with a prothrombotic state and a high incidence of thrombotic event(s) (TE). Objectives: To study platelet reactivity in hospitalized COVID-19 patients and determine a possible association with the clinical outcomes thrombosis and all-cause mortality. Methods: Seventy nine hospitalized COVID-19 patients were enrolled in this retrospective cohort study and provided blood samples in which platelet reactivity in response to stimulation with ADP and TRAP-6 was determined using flow cytometry. Clinical outcomes included thrombotic events, and all-cause mortality. Results: The incidence of TE in this study was 28% and all-cause mortality 16%. Patients that developed a TE were younger than patients that did not develop a TE [median age of 55 vs. 70 years; adjusted odds ratio (AOR) = 0.96 per 1 year of age, 95% confidence interval (CI) 0.92-1.00; p = 0.041]. Furthermore, patients using preexisting thromboprophylaxis were less likely to develop a thrombotic complication than patients that were not (18 vs. 54%; AOR = 0.19, 95% CI 0.04-0.84; p = 0.029). Conversely, having asthma strongly increased the risk on TE development (AOR = 6.2, 95% CI 1.15-33.7; p = 0.034). No significant differences in baseline P-selectin expression or platelet reactivity were observed between the COVID-19 positive patients (n = 79) and COVID-19 negative hospitalized control patients (n = 21), nor between COVID-19 positive survivors or non-survivors. However, patients showed decreased platelet reactivity in response to TRAP-6 following TE development. Conclusion: We observed an association between the use of preexisting thromboprophylaxis and a decreased risk of TE during COVID-19. This suggests that these therapies are beneficial for coping with COVID-19 associated hypercoagulability. This highlights the importance of patient therapy adherence. We observed lowered platelet reactivity after the development of TE, which might be attributed to platelet desensitization during thromboinflammation.

Refractory neutrophils and monocytes in patients with inflammatory bowel disease after repeated bouts of prolonged exercise.

BACKGROUND: Neutrophils and monocytes are key immune effector cells in inflammatory bowel disease (IBD) that is associated with chronic inflammation in the gut. Patients with stable IBD who perform exercise have significantly fewer flare-ups of the disease, but no underlying mechanism has been identified. Therefore, the aim of this study was to compare the responsiveness/refractoriness of these innate immune cells after repeated bouts of prolonged exercise in IBD patients and controls. METHODS: Patients with IBD and age- and gender-matched healthy controls were recruited from a cohort of walkers participating in a 4-day walking event. Blood analysis was performed at baseline and after 3 days of walking. Responsiveness to the bacterial/mitochondrial-stimulus N-Formylmethionine-leucyl-phenylalanine (fMLF) was tested in granulocytes and monocytes by measuring the expression of activation markers after adding this stimulus to whole blood. RESULTS: In total 38 participants (54 ± 12 years) were included in this study: 19 walkers with and 19 walkers without IBD. After 3 days of prolonged exercise, a significant increase in responsiveness to fMLF was observed in all participants irrespective of disease. However, IBD patients showed significantly less responsiveness in neutrophils and monocytes, compared with non-IBD walkers. CONCLUSIONS: Increased responsiveness of neutrophils and monocyte to fMLF was demonstrated after repetitive bouts of prolonged exercise. Interestingly, this exercise was associated with relative refractoriness of both neutrophils and monocytes in IBD patients. These refractory cells might create a lower inflammatory state in the intestine providing a putative mechanism for the decrease in flare-ups in IBD patients after repeated exercise.

Splenectomy is associated with altered leukocyte kinetics after severe trauma.

BACKGROUND: Inadequate activation of the innate immune system after trauma can lead to severe complications such as Acute Respiratory Distress Syndrome and Multiple Organ Dysfunction Syndrome. The spleen is thought to modulate the cellular immune system. Furthermore, splenectomy is associated with improved outcome in severely injured trauma patients. We hypothesized that a splenectomy alters the cellular immune response in polytrauma. METHODS: All adult patients with an ISS ≥ 16 and suffering from splenic or hepatic injuries were selected from our prospective trauma database. Absolute leukocyte numbers in peripheral blood were measured. White blood cell kinetics during the first 14 days were compared between splenectomized patients, patients treated surgically for liver trauma and nonoperatively treated individuals. RESULTS: A total of 129 patients with a mean ISS of 29 were included. Admission characteristics and leukocyte numbers were similar in all groups, except for slightly impaired hemodynamic status in patients with operatively treated liver injuries. On admission, leukocytosis occurred in all groups. During the first 24 h, leukopenia developed gradually, although significantly faster in the operatively treated patients. Thereafter, leukocyte levels normalized in all nonoperatively treated cases whereas leukocytosis persisted in operatively treated patients. This effect was significantly more prominent in splenectomized patients than all other conditions. CONCLUSIONS: This study demonstrates that surgery for intra-abdominal injuries is associated with an early drop in leucocyte numbers in peripheral blood. Moreover, splenectomy in severely injured patients is associated with an altered cellular immune response reflected by a persistent state of prominent leukocytosis after trauma.

Flow cytometric evaluation of the neutrophil compartment in COVID-19 at hospital presentation: A normal response to an abnormal situation.

Coronavirus disease 2019 (COVID-19) is a rapidly emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Critical COVID-19 is thought to be associated with a hyper-inflammatory process that can develop into acute respiratory distress syndrome, a critical disease normally mediated by dysfunctional neutrophils. This study tested the hypothesis whether the neutrophil compartment displays characteristics of hyperinflammation in COVID-19 patients. Therefore, a prospective study was performed on all patients with suspected COVID-19 presenting at the emergency room of a large academic hospital. Blood drawn within 2 d after hospital presentation was analyzed by point-of-care automated flow cytometry and compared with blood samples collected at later time points. COVID-19 patients did not exhibit neutrophilia or eosinopenia. Unexpectedly neutrophil activation markers (CD11b, CD16, CD10, and CD62L) did not differ between COVID-19-positive patients and COVID-19-negative patients diagnosed with other bacterial/viral infections, or between COVID-19 severity groups. In all patients, a decrease was found in the neutrophil maturation markers indicating an inflammation-induced left shift of the neutrophil compartment. In COVID-19 this was associated with disease severity.

An increase in CD62Ldim neutrophils precedes the development of pulmonary embolisms in COVID-19 patients.

OBJECTIVES: A high incidence of pulmonary embolism (PE) is reported in patients with critical coronavirus disease 2019 (COVID-19). Neutrophils may contribute to this through a process referred to as immunothrombosis. The aim of this study was to investigate the occurrence of neutrophil subpopulations in blood preceding the development of COVID-19 associated PE. METHODS: We studied COVID-19 patients admitted to the ICU of our tertiary hospital between 19-03-2020 and 17-05-2020. Point-of-care fully automated flow cytometry was performed prior to ICU admission, measuring the neutrophil activation/maturation markers CD10, CD11b, CD16 and CD62L. Neutrophil receptor expression was compared between patients who did or did not develop PE (as diagnosed on CT angiography) during or after their ICU stay. RESULTS: Among 25 eligible ICU patients, 22 subjects were included for analysis, of whom nine developed PE. The median (IQR) time between neutrophil phenotyping and PE occurrence was 9 (7-12) days. A significant increase in the immune-suppressive neutrophil phenotype CD16bright /CD62Ldim was observed on the day of ICU admission (P = 0.014) in patients developing PE compared to patients who did not. CONCLUSION: The increase in this neutrophil phenotype indicates that the increased number of CD16bright /CD62Ldim neutrophils might be used as prognostic marker to predict those patients that will develop PE in critical COVID-19 patients.

Management of pediatric blunt abdominal trauma in a Dutch level one trauma center.

PURPOSE: Most children with intra-abdominal injuries can be managed non-operatively. However, in Europe, there are many different healthcare systems for the treatment of pediatric trauma patients. Therefore, the aim of this study was to describe the management strategies and outcomes of all pediatric patients with blunt intra-abdominal injuries in our unique dedicated pediatric trauma center with a pediatric trauma surgeon. METHODS: We performed a retrospective, single-center, cohort study to investigate the management of pediatric patients with blunt abdominal trauma. From the National Trauma Registration database, we retrospectively identified pediatric (≤ 18 years) patients with blunt abdominal injuries admitted to the UMCU from January 2012 till January 2018. RESULTS: A total of 121 pediatric patients were included in the study. The median [interquartile range (IQR)] age of patients was 12 (8-16) years, and the median ISS was 16 (9-25). High-grade liver injuries were found in 12 patients. Three patients had a pancreas injury grade V. Furthermore, 2 (1.6%) patients had urethra injuries and 10 (8.2%) hollow viscus injuries were found. Eighteen (14.9%) patients required a laparotomy and 4 (3.3%) patients underwent angiographic embolization. In 6 (5.0%) patients, complications were found and in 4 (3.3%) children intervention was needed for their complication. No mortality was seen in patients treated non-operatively. One patient died in the operative management group. CONCLUSIONS: In conclusion, it is safe to treat most children with blunt abdominal injuries non-operatively if monitoring is adequate. These decisions should be made by the clinicians operating on these children, who should be an integral part of the entire group of treating physicians. Surgical interventions are only needed in case of hemodynamic instability or specific injuries such as bowel perforation.

Analysis of human neutrophil phenotypes as biomarker to monitor exercise-induced immune changes.

The amplitude of the innate immune response reflects the degree of physiological stress imposed by exercise load. An optimal balance of exercise intensity and duration is essential for a balanced immune system and reduces the risk of dysfunction of the immune system. Therefore, it is hypothesized that neutrophils, as key players in the innate immune system, can be used as biomarker in detecting overtraining. The aim was to monitor the state of the innate immune system by phenotyping neutrophils during consecutive bouts of prolonged exercise. Study subjects were recruited from a cohort of walkers participating in a walking event on 3 consecutive days. Participants with immune deficiencies were excluded. Questionnaires to determine the physiological status of the participants were completed. Analysis of neutrophil receptor expression was done by a point-of-care fully automated flow cytometer. A total of 45 participants were recruited, of whom 39 participants were included for data analysis. Study participants had a median age of 64 (58-70) years. The absolute numbers CD16dim /CD62Lbright and CD16bright /CD62Ldim neutrophils were increased after the first 2 days of exercise followed by an adaptation/normalization after the third day. Participants with activated neutrophils (high CD11b expression) had an impaired physical feeling indicated by the participant on a lower visual analog scale compared to participants who did not have activated neutrophils (P = 0.017, P = 0.022). Consecutive days of prolonged exercise results in an initial systemic innate immune response, followed by normalization/adaptation. Increased neutrophil activation was associated with impaired physical feeling measured by a validated VAS score indicated by the participant. Fully automated point-of-care flow cytometry analysis of neutrophil phenotypes in a field laboratory might be a useful tool to monitor relevant differences in the systemic innate immune response in response to exercise.

Predictors of surgical management of high grade blunt splenic injuries in adult trauma patients: a 5-year retrospective cohort study from an academic level I trauma center.

BACKGROUNDS: Splenic injury accounts for 40% of all injuries after blunt abdominal trauma. Blunt splenic injury in hemodynamically unstable patients is preferably treated by splenectomy. Nowadays hemodynamically stable patients with low grade splenic injuries are mostly treated by non-operative management (NOM). However no consensus exists about the management of high grade splenic injuries in hemodynamically stable patients. Therefore the aim of this study was to analyze patients with high grade splenic injuries in our institution. METHODS: We retrospectively included all patients with a splenic injury presented to our level I trauma center during the 5-year period from January 1, 2012, until December 31, 2017. Baseline characteristics, data regarding complications and mortality were collected from the electronic patient registry. Patients were grouped based on splenic injury and the treatment they received. RESULTS: A total of 123 patients were included, of which 93 (75.6%) were male with a median age of 31 (24-52) and a median injury severity score of 27 (17-34). High grade injuries (n = 28) consisted of 20 Grade IV injuries and 8 grade V injuries. Splenectomy was required in 15/28 (53.6%) patients, of whom all remained hemodynamically unstable after resuscitation, including all grade V injuries. A total of 13 patients with high grade injuries were treated with spleen preserving therapy. Seven of these patients received angio-embolization. One patient went for laparotomy and the spleen was treated with a hemostatic agent. Secondary hemorrhage was present in 3 of these patients (initial treatment: 1 embolization/ 2 observational), resulting in a success rate of 76.9%. There is no mortality seen in patient with high grade splenic injuries. CONCLUSION: Non-operative treatment in high grade splenic injuries is a safe treatment modality in hemodynamically stable patients. Hemodynamic status and peroperative bleeding, not injury severity or splenic injury grade were the drivers for surgical management by splenectomy. This selected cohort of patients must be closely monitored to prevent adverse outcomes from secondary delayed bleeding in case of non-operative management.

Point-of-Care Analysis of Neutrophil Phenotypes: A First Step Toward Immuno-Based Precision Medicine in the Trauma ICU.

OBJECTIVES: The amount of tissue damage and the amplitude of the immune response after trauma are related to the development of infectious complications later on. Changes in the neutrophil compartment can be used as read out of the amplitude of the immune response after trauma. The study aim was to test whether 24/7 point-of-care analysis of neutrophil marker expression by automated flow cytometry can be achieved after trauma. DESIGN: A prospective cohort study was performed. Polytrauma patients who developed infectious complications were compared with polytrauma patients who did not develop infectious complications. SETTING: The study was performed in a level 1 trauma center. PATIENTS: All trauma patients presented in the trauma bay were included. INTERVENTIONS: An extra blood tube was drawn from all patients. Thereafter, a member of the trauma team placed the blood tube in the fully automated flow cytometer, which was located in the corner of the trauma room. Next, a modified and tailored protocol for this study was automatically performed. MAIN RESULTS: The trauma team was able to successfully start the point-of-care automated flow cytometry analysis in 156 of 164 patients, resulting in a 95% success rate. Polytrauma patients who developed infectious complications had a significantly higher %CD16dim/CD62Lbright neutrophils compared with polytrauma patients who did not develop infectious complications (p = 0.002). Area under the curve value for %CD16dim/CD62Lbright neutrophils is 0.90 (0.83-0.97). CONCLUSIONS: This study showed the feasibility of the implementation of a fully automated point-of-care flow cytometry system for the characterization of the cellular innate immune response in trauma patients. This study supports the concept that the assessment of CD16dim/CD62Lbright neutrophils can be used for early detection of patients at risk for infectious complications. Furthermore, this can be used as first step toward immuno-based precision medicine of polytrauma patients at the ICU.

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.

New automated analysis to monitor neutrophil function point-of-care in the intensive care unit after trauma.

BACKGROUND: Patients often develop infectious complications after severe trauma. No biomarkers exist that enable early identification of patients who are at risk. Neutrophils are important immune cells that combat these infections by phagocytosis and killing of pathogens. Analysis of neutrophil function used to be laborious and was therefore not applicable in routine diagnostics. Hence, we developed a quick and point-of-care method to assess a critical part of neutrophil function, neutrophil phagosomal acidification. The aim of this study was to investigate whether this method was able to analyze neutrophil functionality in severely injured patients and whether a relation with the development of infectious complications was present. RESULTS: Fifteen severely injured patients (median ISS of 33) were included, of whom 6 developed an infection between day 4 and day 9 after trauma. The injury severity score did not significantly differ between patients who developed an infection and patients who did not (p = 0.529). Patients who developed an infection showed increased acidification immediately after trauma (p = 0.006) and after 3 days (p = 0.026) and a decrease in the days thereafter to levels in the lower normal range. In contrast, patients who did not develop infectious complications showed high-normal acidification within the first days and increased tasset to identify patients at risk for infections after trauma and to monitor the inflammatory state of these trauma patients. CONCLUSION: Neutrophil function can be measured in the ICU setting by rapid point-of-care analysis of phagosomal acidification. This analysis differed between trauma patients who developed infectious complications and trauma patients who did not. Therefore, this assay might prove a valuable asset to identify patients at risk for infections after trauma and to monitor the inflammatory state of these trauma patients. TRIAL REGISTRATION: Central Committee on Research Involving Human Subjects, NL43279.041.13. Registered 14 February 2014. https://www.toetsingonline.nl/to/ccmo_search.nsf/Searchform?OpenForm.

Persistent Inflammation, Immunosuppression and Catabolism Syndrome (PICS) after Polytrauma: A Rare Syndrome with Major Consequences.

Nowadays, more trauma patients develop chronic critical illness (CCI), a state characterized by prolonged intensive care. Some of these CCI patients have disproportional difficulties to recover and suffer from recurrent infections, a syndrome described as the persistent inflammation, immunosuppression and catabolism syndrome (PICS). A total of 78 trauma patients with an ICU stay of ≥14 days (CCI patients) between 2007 and 2017 were retrospectively included. Within this group, PICS patients were identified through two ways: (1) their clinical course (≥3 infectious complications) and (2) by laboratory markers suggested in the literature (C-reactive protein (CRP) and lymphocytes), both in combination with evidence of increased catabolism. The incidence of PICS was 4.7 per 1000 multitrauma patients. The sensitivity and specificity of the laboratory markers was 44% and 73%, respectively. PICS patients had a longer hospital stay (median 83 vs. 40, p < 0.001) and required significantly more surgical interventions (median 13 vs. 3, p = 0.003) than other CCI patients. Thirteen PICS patients developed sepsis (72%) and 12 (67%) were readmitted at least once due to an infection. In conclusion, patients who develop PICS experience recurrent infectious complications that lead to prolonged hospitalization, many surgical procedures and frequent readmissions. Therefore, PICS forms a substantial burden on the patient and the hospital, despite its low incidence.

Nonoperative management of splenic injury in closely monitored patients with reduced consciousness is safe and feasible.

BACKGROUND: Treatment of blunt splenic injury has changed over the past decades. Nonoperative management (NOM) is the treatment of choice. Adequate patient selection is a prerequisite for successful NOM. Impaired mental status is considered as a relative contra indication for NOM. However, the impact of altered consciousness in well-equipped trauma institutes is unclear. We hypothesized that impaired mental status does not affect outcome in patients with splenic trauma. METHODS: Our prospectively composed trauma database was used and adult patients with blunt splenic injury were included during a 14-year time period. Treatment guidelines remained unaltered over time. Patients were grouped based on the presence (Group GCS: < 14) or absence (Group GCS: 14-15) of impaired mental status. Outcome was compared. RESULTS: A total of 161 patients were included, of whom 82 were selected for NOM. 36% of patients had a GCS-score < 14 (N = 20). The median GCS-score in patients with reduced consciousness was 9 (range 6-12). Groups were comparable except for significantly higher injury severity scores in the impaired mental status group (19 vs. 17, p = 0.007). Length of stay (28 vs. 9 days, p < 0.001) and ICU-stay (8 vs. 0 days, p = 0.005) were longer in patients with decreased GCS-scores. Failure of NOM, total splenectomy rates, complications and mortality did not differ between both study groups. CONCLUSION: This study shows that NOM for blunt splenic trauma is a viable treatment modality in well-equipped institutions, regardless of the patients mental status. However, the presence of neurologic impairment is associated with prolonged ICU-stay and hospitalization. We recommend, in institutions with adequate monitoring facilities, to attempt nonoperative management for blunt splenic injury, in all hemodynamically stable patients without hollow organ injuries, also in the case of reduced consciousness.

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.