Shock, Publish Ahead of Print DOI : 10.1097/SHK.0000000000001027 Value of the delta neutrophil index for predicting 28-day mortality in patients with acute pulmonary embolism in the emergency department Taeyoung Kong 1, YooSeok Park 1, Hye Sun Lee 2, Sinae Kim2, Jong Wook Lee3,4,Gina Yu 1, Claire Eun5, Je Sung You 1*, Hyun Soo Chung1, Incheol Park 1, Sung Phil Chung1 1 Department of Emergency Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea 2 Department of Research Affairs, Biostatistics Collaboration Unit, Yonsei University College of Medicine, Seoul, Republic of Korea 3 Department of Laboratory Medicine, Konyang University Hospital, Daejeon, Republic of Korea 4 Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea 5 Department of Neurology, University of California, San Francisco, and the San Francisco Veterans Affairs Medical Center, San Francisco, CA, United States Corresponding author: Je Sung You, MD, PhD* Department of Emergency Medicine, Yonsei University College of Medicine, 211 Eonju-Ro, Gangnam-Gu, Seoul 135-720, Republic of Korea E-mail: firstname.lastname@example.org, Tel: +82-2-2019-3030, Fax: +82-2-2019-4820 Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Running head:Delta neutrophil index in acute pulmonary embolism Word Counts: Abstract = 250, Main Text = 4640 Declaration of Interests The authors declare no conflict of interests Disclosure of funding J. S. You received support from the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2015R1C1A1A01054641), and the Yonsei University Future-leading Research Initiative for 2015 (2015-22-0096). S.P. Chung and T.Y. Kong were supported by basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2017R1A2B4012378). J.S.You received research fund from Siemens Health Care. However, the fund did not exceed $10,000/year. The funding bodies had no role in the design, collection, analysis, or interpretation of this study. The other authors have no financial conflicts of interest. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. ABSTRACT Purpose: Acute pulmonary embolism (PE), frequently seen in the emergency department (ED), is a leading cause of cardiovascular morbidity and mortality. The delta neutrophil index (DNI) reflects the fraction of circulating immature granulocytes as a component of the systemic inflammatory response syndrome criteria. The pathogenesis of acute PE is significantly associated with inflammation.The aim of the study was to investigate the clinical usefulness of the DNI as a marker of severity in patients with acute PE admitted to the ED. Methods: We retrospectively analysed the data of patients who were diagnosed with acute PEat a single ED, admitted from 1 January 2011 to 30 June 2017. The diagnosis of acute pulmonary embolism was confirmed using clinical, laboratory, and radiological findings. The DNI was determined at presentation. The clinical outcome was all-cause mortality within 28 days of emergency departmentadmission. Results: We included 447 patients in this study. The multivariate Cox regression model demonstrated that higher DNI values on EDadmission were significantly associated with short-term mortality (hazard ratio, 1.107; 95% confidence interval, 1.042–1.177). The optimal cut-off DNI value, measured on EDadmission, was 3.0%; this value was associated with an increased hazard of 28-day mortality following PE(HR, 7.447; 95% CI, 4.183–13.366; p < 0.001) Conclusion: The DNI value, obtained as part of the complete blood count analysis, can be easily determined without additional burdens of cost or time. A high DNI is useful as a marker to predict 28day mortality in patients with acute PE. Keywords: immature granulocyte; inflammation; mortality; prognostication; pulmonary embolism; shock; sterile Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. INTRODUCTION Acute pulmonary embolism (PE) is a serious clinical condition and a leading cause of cardiovascular hospitalisation, morbidity, and mortality (1-3). Although acute PE may be asymptomatic, being incidentally diagnosed during imaging, the International Cooperative Pulmonary Embolism Registry has reported fatality rates of approximately 15% and 58% in haemodynamically stable and unstable patients, respectively (1). Acute PE has a high 28-day mortality rate of 9–31% (4). The severity of acute PE is classified according to the probability of early death (2). Right ventricular (RV) dysfunction and failure resulting from acute pressure overload are the major determinants of a patient’s early clinical course and risk of an adverse outcome (2, 5). Although most patients with acute PE are normotensive in the early stage, some develop shock despite receiving appropriate treatment (6). It is important that acute PE risk stratification is optimal on admission to the emergency department (ED), before sustained hypotension develops. In addition, early identification of patients at high risk of a poor outcome may improve the survival rate of patients with acute PE. However, it is difficult to ascertain risk in an emergency situation. Hence, many studies have attempted to characterize risk factors and comorbidities in patients by using a high index of suspicion for acute PE in the early stage of disease (1, 7, 8). The Pulmonary Embolism Severity Index (PESI) is a useful, practical clinical prediction rule. It uses 11 predictors from the patient’s medical history and physical examination (9). The PESI has discriminative power to predict short-term mortality and adverse outcomes in patients with acute PE (5, 9) and may be suitable for clinical application. However, the main limitation of the PESI is that it requires numerous variables and is relatively complex to calculate (9). Therefore, there is a need for new prognostic markers that can be measured rapidly and readily in the ED. Acute PE is significantly associated with vascular inflammation in the pulmonary arteries, pulmonary inflammation as a result of pulmonary infarction, and myocyte damage and RV dysfunction due to cardiac inflammation (8). The sustained, exacerbated inflammatory response is strongly associated with Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. increased mortality (8, 10, 11). Many studies have been conducted on the association between predictors of severity of acute PE and inflammation-related markers (7, 8, 10-14). Immature granulocytes have been used as a marker of inflammation, but this has been regarded as being more important in the context of local infection, sepsis, and septic shock (15-18). There are some major limitations to the measurement of immature granulocytes, including difficulty in obtaining a rapid manual measurement from examination of a stained blood smear and possible inaccuracy when using only a 200-cell manual differential count method (15, 19). The delta neutrophil index (DNI)reflects myeloperoxidase (MPO)-reactive cells lacking nuclear lobularity as polymorphonuclear myeloidderived suppressor cells, and they may reflectthe fraction of circulating immature granulocytes, including metamyelocytes, myelocytes, and promyelocytes as the leukocyte subfraction.(17, 19) (Appendix 1,http://links.lww.com/SHK/A654)Nahm et al. demonstrated that the DNI correlated strongly with manual immature granulocyte count (r = 0.75) (19). As a recent technological advance, the specific automated blood cell analyser can rapidly and easily determine the DNI while determining the complete blood count (CBC) (16, 17, 19). The DNI is measured by leukocyte differentials, with two independent channels, using flow cytometric principles (17, 19). It iscalculated according to the following formula: DNI = (neutrophil sub-fraction + eosinophil sub-fraction measured in the myeloperoxidase channel) − (polymorphonuclear [PMN] sub-fraction measured in the nuclear lobularity channel) (17, 19-21). A strong association has been reported between DNI and increased mortality in patients with systemic sterile inflammation, such as those with out-of-hospital cardiac arrest and upper gastrointestinal haemorrhage (20, 22). The aim of the present study was to evaluate whether an increased DNI is able to predict 28-day mortality in patients with acute PE, and to investigate the clinical usefulness of the DNI as a marker of severity in patients with acute PE admitted to the ED. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. PATIENTS AND METHODS Study population This retrospective observational study was performed in the ED of Severance Hospital, a universityaffiliated, tertiary level referral hospital with an annual census of approximately 85,000 visits. The institutional review board of Yonsei University Health System (No 3-2017-0036) reviewed and approved the study. The requirement for written informed consent from patients was waived. We retrospectively identified consecutive adult patients (>18 years old) with acute PE admitted to the ED from 1 January 2011 to 30 June 2017. We retrospectively analysed the data of patients who were diagnosed with acute PE at a single ED admission and had a final diagnosis of acute PE (according to ICD-9 codes: I.260/ I.269) based on the findings of computed tomography (CT) performed in the ED. The definitive CT-based diagnosis of acute PE was defined as the presence of at least one intraluminal filling defect in an interlobar or more proximal pulmonary artery (6). We also analysed the patients’ electronic medical records. The enrolment, exclusion, and clinical outcome data for patients with acute PE are shown in Figure 1. We excluded patients with acute myocardial infarction, those who received chemotherapy within 7 days prior to ED admission, those with concurrent infection at the time of ED admission, those transferred out to other hospitals, and those who self-discharged against medical advice. Other exclusion criteria were chronic inflammatory disorders, including autoimmune diseases, a history of previous or current haematological malignancy, and chronic PE. The primary end-point of this study was all-cause mortality within 28 days of ED admission following acute PE. In addition, the secondary end point was in-hospital development of hypotension or shock. To investigate in-hospital occurrence of hypotension or shock, in additional analysis, we excluded if they had hypotension (as defined by systolic blood pressure (SBP) <90 mmHg, shock or the need for intravenous infusion of catecholamine) on ED admission and the need for ventilator support (n=32)(6). In-hospital occurrence of hypotension was defined as SBP less than 90 mmHg for more than 15 min without signs of hypovolemia (bleeding, dehydration, vomiting, diarrhea, adverse effects of drugs) or sepsis, within 24 h Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. of admission and In-hospital development of shock was defined as patients with arterial hypotension accompanied by cardiogenic shock or judged by the attending physicians to require the administration of catecholamines(6). Data collection We collected data on demographic characteristics (age; sex; previous medical history, including previous PE, previous deep vein thrombosis, and malignancy); health-related complaints; haemodynamic parameters; laboratory results; radiological findings, including involved artery; echocardiographic findings, including RV dilatation; in-hospital course and clinical outcome; the main type of treatment received;and period of follow-up. The DNI for each patient was determined using venous blood in EDTA (ethylenediaminetetraacetic) containing vacutainerson presentation to the ED (time-0; within 15 minutesafter ED admission) andtime-24 (24 ± 6 hours after admission). To assess the DNI, we used the same type of haematology analyser (ADVIA 2120; Siemens, Forchheim, Germany) used for the analysis of the CBC. The PESI was measured on ED admission to evaluate the clinical severity of each patient. DNI measurement The specific analysers used comprise an optical system based on a cytochemical myeloperoxidase tungsten-halogen channel (that measures and differentiates neutrophils, eosinophils, lymphocytes, monocytes, and large unstained cells based on size and myeloperoxidase staining intensity) and a laserdiode channel (that calculates, classifies, and counts cell types with respect to lobularity/nuclear density and size)(15, 19, 20, 22). The DNI was then calculated by subtracting the fraction of mature PMNs from the sum of the myeloperoxidase-reactive cells, detecting circulating immature granulocytes as the leukocyte sub-fraction (15, 19, 20, 22). We also performed other laboratory tests, including determination of cardiac markers, electrolytes, D-dimer, high sensitivity C-reactive protein (hs-CRP) and creatinine at the time of ED admission. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Statistical analysis We presented demographic and clinical data as the median and interquartile range (IQR), mean ± standard deviation (SD), or percentage and frequency, as appropriate. We compared continuous variables using a two-sample t-test or the Mann–Whitney U-test and categorical variables using the χ2 test or Fisher’s exact test. A linear mixed model and repeated measures covariance pattern with unstructured covariance within patients were estimated to assess significant differences between survival and non-survival groups over time. Two fixed effects were assessed: A time effect (time: DNI obtained on admission and 24-h after ED admission) and a clinical effect (level: survival and nonsurvival on 28-day). Differences in clinical effect over time were analysed in accordance with clinical effect × time effect. In addition, we also measured significant differences between groups over time for the development of in-hospital hypotension.We determined the area under the curve (AUC) using receiver operating characteristic curves (ROC) to identify the effect of the DNI for predicting the occurrence of in-hospital hypotension and 28-day mortality in patients with acute PE. Youden’s method was used to determine the optimal DNI cut-off value. In addition, we combined the DNI on ED admission with the PESI, using an ROC analysis. Univariable Cox proportional hazards regression analyses were conducted to assess relationships between demographic characteristics and clinical data. A multivariable Cox proportional hazards regression analysis that integrated the major covariates (variables with a p< 0.05) identified in the univariable analyses was also performed to identify promising independent factors predictive of 28-day mortality, considering time-to-event data in patients with PE. We used the PESI as a variable in this model because this scoring system, which was created by integrating the clinically significant variables identified in this study, can represent various variables. The results are expressed as hazard ratios (HRs) and 95% confidence intervals (CIs). In this study, we used the calibration plot to confirm the suitability of the applied prediction model. We combined the variables included in the multivariable Cox proportional hazard and calculated the probability for occurrence of an event. We created KaplanMeier survival curves using 28-day mortality data, and compared groups using the log-rank test. We Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. calculated Harrell’s C-index to determine the time-dependent discriminatory ability and ability of DNI to predict 28-day mortality in patients with acute PE (23). To determine the additional predictive power of the DNI, we compared it with the C index to assess whether the DNI provided the better prognostic value. In addition, the PESI is a useful scoring system to predict mortality in patients with acute PE. To identify improvement of the predictive power of the DNI for 28-day mortality, we compared the change in AUC, the integrated discrimination improvement (IDI), and continuous net reclassification improvement (NRI) values between the PESI and PESI+DNI.Although previous studies estimated cutoff values based only on events, we also estimated optimal cut-off values for the dichotomization of the clinical outcome variable based on time-to-event data using the technique devised by Contal and O’Quigley(16, 20). We selected the optimal cut-off point by maximizing the HR. To identify the exact cut-off point, we determined the cut-off point in the discovery cohort by using the Contal and O’Quigley method, after which we performed internal validation using bootstrapping (a mean HR of 1,000 repetitions). We performed internal validation with >3.0% of the optimal cut-off points and calculated HR, accuracy, sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) to predict 28-day mortality. Statistical analyses were performed using SAS software, version 9.2 (SAS Institute Inc., Cary, NC); R software, version 3.2.5 for Windows (the R foundation for statistical computing, Vienna, Austria; http://www.R-project.org/); and MedCalc, version 12.7.0 (MedCalc Software, Ostend, Belgium). A p-value < 0.05 was considered significant. RESULTS A total of 447patients were included in this study. The enrolment, exclusions, and clinical outcome data for patients with acute PE are shown in Figure 1. Of 447 study patients, 46 died within 28 days. Of 46 deaths, 7 patients had chest pain and 5 had haemoptysis. There was no significant difference in the development of chest pain between the survival (20.7%) and non-survival group (15.2%, p=0.38). However, the development of haemoptysis was significantly higher in the mortality group (10.87%) than in the survival group (2.99%, p=0.022). There were significant differences in DNI values on ED Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. admission between the survival (1.16%) and non-survival (4.41%) groups according to 28-day mortality (p< 0.001) (Table 1 and appendix 2, http://links.lww.com/SHK/A654). The mean PESI score was significantly higher in the non-survival (125.9± 38.4) than in the survival (102± 37) group (p< 0.001). Of 415 study patients, 44 (10.6%) developed the hypotension or shock. Figures showed effects obtained for time, by group, and by interaction according to 28-day mortality and the development of hypotension or shock (Fig. 2A, B and appendix 3, http://links.lww.com/SHK/A654).The AUC for the DNI on ED admission for predicting 28-day mortality was 0.767 (p< 0.001) (Fig. 2C). When the PESI was combined with DNI on ED admission in the ROC analysis, the AUC increased significantly from 0.695 to 0.809 (p< 0.001) (Fig. 3A and appendix 4, http://links.lww.com/SHK/A654). In the prediction of 28-day mortality, when the DNI at time-0 was added to the PESI, the change in the AUC was 0.114 (range, 0.051–0.177, p=0.001), NRI was 0.104 (range, 0.044–0.163, p=0.001), and IDI was 0.337 (range, 0.16–0.514, p=0.04). Thus, the predictive power of DNI for 28-day mortality improved. When the DNI at time-0 was added to the PESI, the change in the AUC, IDI, and NRI values were higher than that with the addition of the WBC count at time-0 to the PESI (Appendix 5, http://links.lww.com/SHK/A654). We demonstrated that the DNI on ED admission in the univariable Cox regression analyses differed significantly between the survival and non-survival groups, stratified by 28-day mortality (Appendix 6 and 7, http://links.lww.com/SHK/A654). The multivariable Cox regression model demonstrated that the DNI on ED admission (HR, 1.107; 95% CI, 1.042–1.177; p = 0.001) was a significant independent predictor of 28-day mortality in patients with acute PE (Table 2). To improve the statistical reliability, all variables that were significant (p<0.05) in the univariable analysis were included in the multivariable Cox proportional hazard model of this study. The multivariable Cox proportional hazard model, including all these variables, further confirmed the significant association between increased DNI values at time-0 and time-24 and an increased risk of 28-day mortality and development of shock or hypotension among patients with PE (Appendices8, 9, and 10, http://links.lww.com/SHK/A654).We confirmed that this probability is consistent with the occurrence rate of actual events using the Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. calibration plot (Appendix 11, http://links.lww.com/SHK/A654).The Kaplan–Meier curves (Fig. 3B) demonstrated that the DNI value on ED admission independently predicted 28-day mortality in patients with acute PE (p < 0.001). The optimal cut-off of DNI for predicting 28-day mortality was 3.0% at ED admission (p < 0.001(sensitivity: 54.35% [39–69.1]; specificity: 88.78% [85.3–91.7])); a DNI >3.0% at ED admission was strongly associated with an increased riskof short-term mortality among patients with acute PE (HR, 7.447; 95% CI, 4.183–13.366; p< 0.001) (Appendix 12, http://links.lww.com/SHK/A654). We performed internal validation using bootstrapping (a mean HR of 1,000 repetitions). The results obtained by internal validation were similar to those of the deviation cohort (Table 3).The Harrell’s C-index of DNI on ED admission for predicting 28-day mortality was 0.752 (95% CI 0.673–0.82, p< 0.001). The white blood cell (WBC) count, platelet count, and haemoglobin level can also be determined automatically along with the CBC. The C-statistic of the DNI on ED admission was statistically superior to that of the WBC count, platelet count, and haemoglobin level on ED admission for predicting 28-day mortality. Compared with the Harrell Cindex of the present study, the C-statistic of the DNI on ED admission was not statistically inferior to that of the PESI score and hs-CRP level for predicting 28-day mortality. In addition, the C-statistic of the DNI on ED admission was statistically superior to that of the troponin I and D-dimer level for predicting 28-day mortality (Fig. 4 and appendix 13,http://links.lww.com/SHK/A654). DISCUSSION To the best of our knowledge, this is the first study to determine the usefulness of the DNI, reflecting the number of circulating immature granulocytes, to predict short-term mortality in patients with acute PE in an emergency setting. Acute PE is generally a critical condition that leads to death soon after ED admission (1). Risk stratification should be promptly conducted in the ED (1). In the present study, we demonstrated that DNI was a significant independent predictor of 28-day mortality in patients with acute PE. We determined that DNI values >3.0% on ED admission could significantly predict 28-day mortality in this group of patients. Hence, these DNI values—obtained rapidly, easily, and inexpensively as part of the CBC measurement—can be used to assess severity regardless of Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. haemodynamic instability in patients with acute PE (15, 22, 24). Predicting the prognosis of patients with acute PE has been based on clinical features and markers reflecting myocardial injury or dysfunction (1). In haemodynamically stable patients, RV hypokinesis and dilatation measured by echocardiography or RV dysfunction assessed by multi-detector CT are well-known independent predictors of short-term mortality (1, 25, 26). In the present study, age, systolic blood pressure, white blood cell count, haemoglobin level, NT-pro BNP, D-dimer, hs-CRP, t-CO2, RV dilatation, and the PESI (including age; sex; a history of cancer, heart failure, and chronic lung disease; heart rate ≥110/min; systolic blood pressure <110 mm Hg; respiratory rate ≥30/min; temperature <36 °C; alerted mental status; and oxygen saturation <90%) were risk factors for 28-day mortality among patients with acute PE. Although many studies have attempted to stratify risk in patients with acute PE, simple and readily available markers to assess prognosis are still needed in the emergency setting (8). Despite the relative complexity of using 11 predictors to calculate the PESI in the emergency setting, the PESI is based only on variables related to the medical history and physical examination; it does not require laboratory evaluation (9). Although this is a benefit, it does mean that the score simply reflects the general and current condition of a patient, it might not reflect the severity of a specific disease (27). Moreover, variability in even one or two parameters (such as age or sex) may lead to the classification of patients as being high- or low-risk (27). Using ROC analysis, we demonstrated that the PESI combined with DNI on ED admission improved its discriminative power to predict 28-day mortality. In acute PE, a higher value of troponin is strongly correlated with RV dysfunction as marker of cardiomyocyte damage (6). NT-proBNP, a stress-related marker released by myocytes, is elevated in patients with adverse outcomes after acute PE (28, 29). A systemic review by Sanchez et al. demonstrated that the unadjusted relative risk to predict in-hospital or 30-day mortality was 8.3 (95% CI 3.6–19.3) for troponin-T, 9.5 (95% CI 3.1–28.6) for brain natriuretic peptide, and 5.7 (95% CI 2.2– 15.1) for NT-proBNP(26). Higher values of cardiac markers were significantly associated with an increased risk of short-term mortality in haemodynamically stable patients with acute PE (1, 26). In the present study, we also compared the Harrell’s C-index of PESI score, D-dimer, and troponin-I for Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. predicting 28-day mortality. The C-statistic of DNI at ED admission was not statistically inferior to that of PESI score and hs-CRPfor predicting 28-day mortality. The C-statistic of the DNI on ED admission was statistically superior to that of the troponin I and D-dimer level for predicting 28-day mortality. Considering the easy availability and cost-effectiveness of the DNI compared with the measurement of natriuretic peptides, troponins, and C-reactive protein, DNI may represent a valuable alternative marker for risk stratification in patients with PE.In addition, PE may be completely asymptomatic and be discovered incidentally during diagnostic work-up for another disease or at autopsy(30).The DNI at time 0 may not reflect the exactduration between the onset of symptoms and measurement of the DNI on ED admission.As a result, this suggests the possibility that lower DNI values in some patients were assessed because the patients presented too early for an increase in the DNI to be apparent. The present study demonstrated a significant association between increased DNI values at time 0 and time 24 and an increased risk of 28-day mortality and the development of shock or hypotension among patients with PE. Serial DNI measurements should be considered because the DNI value can be easily determined without additional burdens of cost or time. Although the pathophysiological mechanisms by which PE induces severe morbidity and mortality are not completely understood, Virchow’s triad (blood flow alteration, damage to the vessel wall, and hypercoagulability) is considered the main mechanism (31). However, inflammation (including that occurring as a result of ischaemia, pulmonary arterial hypertension, and thrombus-endothelial interaction) also plays an important role (8). Abul et al. demonstrated that in patients with PE, the CRP (a well-known marker of inflammation and tissue damage) is strongly associated with RV dysfunction and mortality at 36 months (8). Intravascular healing processes are activated by damage-associated molecular patterns of non-infectious cellular debris that cause the release of several immune components, such as cytokines, chemokines, and several types of leukocytes. These contribute to the inflammatory responses of PE as a double-edged sword: The impaired thrombosis resolves, but the risk of PE-related complications increases (31-33). Inflammation following PE contributes to damage and dysfunction of the RV and to cardiac inflammation (8, 14, 34). In addition, vascular inflammatory Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. reactions and pulmonary parenchymal inflammation can be induced by PE (12). Despite the benefit of thrombus resolution by late activation of the neutrophil population, neutrophils are significantly associated with acute severe damage to the lung parenchyma and the RV of the heart in the early stage of the acute inflammatory response following acute PE (14, 31). There is a significant increase in PMN infiltration into the alveolar cavity; this damages the tissues (12). Watts et al. demonstrated that the influx of neutrophils in the inflammatory phase starts 8–18 h after PE (34). Changes occur in the RV within 24 h of PE; the colour changes to white and there is increased myeloperoxidase activity, indicating the presence and activation of PMNs in the RV (14, 31, 34). Given the importance of neutrophils in the pathogenesis of acute PE, several studies have suggested mechanisms to explain this early and rapid release of immature granulocytes. In patients with upper gastrointestinal haemorrhage (UGIH),Kong et al. proposed that massive bleeding at the injured site preferentially induces rapid expansion of circulating neutrophils to compensate for the loss of active neutrophils secondary to the massive loss, consumption, and destruction of mature cells(22, 35). Massive haemorrhage or shock is associated with the production of proinflammatory cytokines and chemokines(22, 36). In the pathogenesis of acute PE,First, the haematopoietic system is rapidly able to switch from steady-state to emergency granulopoiesis to compensate for the loss of active neutrophils secondary to massive consumption resulting from neutrophil infiltration and from the destruction of mature cells under stress conditions such as severe infection and hypoperfusion(34, 36, 37). The increased production of pro-inflammatory cytokines and chemokines (such as interleukin (IL)-6, IL-8, and tumour necrosis factor-alpha) induces rapid expansion of neutrophils soon after PE. This exacerbates the local and systemic inflammatory response. However, severe systemic and sterile inflammation can result in microvascular dysfunction, tissue damage, and dysregulation of metabolism (36). Second, widespread inflammation requires a profound ‘compensatory’ downregulation of immune responses (37). Neutrophil paralysis—known as dysregulated neutrophil function—attenuates tissue damage in severe sterile inflammation as a result of impaired migration of neutrophils to the injured site and neutrophil sequestration in remote organs (37, 38). Consequently, the number of circulating Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. immature granulocytes may increase to compensate for the rapid decrease in the number of active neutrophils. Under these conditions, the host is highly susceptible to infections. Moreover, dysregulation of immune mechanisms may increase mortality (37). Third, sustained hypotension and shock as a result of overt RV failure are significantly associated with higher mortality in patients with acute PE; hence, immediate reperfusion therapy is needed (1, 2). Sauneuf et al. proposed bone marrow exhaustion as a further mechanism by which severe ischaemia-induced inflammation can lead to transient failure in the regulation of neutrophil release during ischaemia and following resuscitation (39, 40). In particular, haemodynamic instability or persistent severe inflammation due to an increase in the severity of acute PE may affect critical regulatory mechanisms for neutrophil release from the bone marrow. Our results suggest that patients with acute PE should be carefully monitored if the DNI value exceeds 3.0%, considering the association between this value and 28-day mortality (HR, 7.477; 95% CI, 4.183–13.366; p < 0.001). A previous study by Kong et al. reported that the optimal cut-off values for DNI on ED admission and on day 1 were 1% (HR, 4.09) and 2.6% (HR, 7.85) and that these levels were associated with an increased hazard of 30-day mortality following upper gastrointestinal bleeding (22). Regarding the severity of disease, Yune et al. demonstrated that DNI values >8.4% on admission (HR, 3.227) and DNI >10.5% on day 1 (HR, 3.292) were associated with increased 30-day mortality in patients surviving out-of-hospital cardiac arrest (20). These findings imply that a higher DNI reflects greater severity of systemic and sterile inflammation and of the disease process (20).Considering mechanisms for this early and rapid release of immature granulocytes,an increased DNI may not be solely specific to pulmonary embolism-related mortality. Therefore, the DNI predicts the mortality for severe diseases associated with severe inflammation, reflecting increased pro-inflammatory cytokines and other mediators. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Study limitations This study has some limitations. First, it was a single-centre retrospective study, increasing the possibility of selection bias. Large, multi-centre, prospective studies and randomized clinical trials are required to validate the clinical usefulness of the DNI as a prognostic marker in patients with acute PE. Second, we assessed only short-term mortality in patients with PE; the long-term clinical outcomes also need to be assessed. Third, although the troponin, D-dimer, and hs-CRP levels in most patients were obtained at time-0, these values at time-24 were not mandatorily determined in patients with acute PE to ensure cost-effectiveness. In this study, we could not directly compare the predictability of clinical outcomes with values at time-0 and time-24 for the troponin, D-dimer, and hs-CRP levels.However, the DNI, as a promising predictor of acute PE, has the additional benefit of being automatically analyzed with the CBC, which is routinely and immediately performed in critically ill patients, without additional time or costs required. Considering the disease severity of PE, the CBC test is usuallyperformed serially in the intensive care unit. In this situation, an increased DNI is able to predict severity in patients with acute PE, and the DNI as a marker of severity canhelp clinicians consider more specific markers with a higher cost. Fourth, although we excluded patients with infectious conditions within 24 h of ED admission a priori(41), the production of immature granulocytes by the bone marrow may be influenced by infection, in addition to stress and systemic inflammation. Further studies are needed to identify the clinical effects of the DNI in patients with infection and systemic sterile inflammation. Finally, although studies have investigated the effects of systemic inflammation, we were unable to evaluate measures of the inflammatory response (such as pro-inflammatory cytokines and chemokines) and to compare the DNI with such inflammatory markers. Further studies are required to compare directly DNI values and indicators of the severity of systemic inflammation as prognostic markers in patients with acute PE. In addition, the pathophysiological mechanisms by which acute PE induces severe morbidity and mortality are not completely understood; therefore, further molecular studies are needed to validate the direct effects of immature granulocytes on the progression of acute PE. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. CONCLUSION The DNI values obtained as part of the CBC can be easily determined with no additional cost or time burden. An increased DNI value is useful as a marker to predict 28-day mortality in patients with acute PE. Declaration of Interests The authors declare no conflict of interests Disclosure of funding J. S. You received support from the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2015R1C1A1A01054641), and the Yonsei University Future-leading Research Initiative for 2015 (2015-22-0096). S.P. Chung and T.Y. Kong were supported by basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2017R1A2B4012378). J.S.You received research fund from Siemens Health Care. However, the fund did not exceed $10,000/year. The funding bodies had no role in the design, collection, analysis, or interpretation of this study. The other authors have no financial conflicts of interest. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. REFERENCES 1. Agnelli G, Becattini C: Acute pulmonary embolism. N Engl J Med 363(3):266-274, 2010. 2. 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Figure legends Figure 1.Flow diagram of patient enrolment, exclusions, and clinical outcomes. PE, pulmonary embolism; ED, emergency medicine; CT, computed tomography; DNAR, do not attempt resuscitation Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Figure 2. Linear mixed model of the DNI to estimate significant differences between groups over time according to 28-day mortality (A) and the occurrence of in-hospital hypotension or shock (B); receiver operating characteristic curves for the predictability of the DNI on ED admission for 28-day mortality (C) and the occurrence of in-hospital hypotension or shock(D). DNI, delta neutrophil index; AUC, area under the curve; ED, emergency department Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Figure 3. (A) A combination of the DNI and the PESI significantly improves the area under the curve compared to the PESI alone; (B) based on the Kaplan-Meier curves for 28-day mortality, results of the log-rank tests demonstrate that a higher DNI is an independent risk factor for patients with acute pulmonary embolism. DNI, delta neutrophil index; PESI, Pulmonary Embolism Severity Index Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Figure 4. Comparison of Harrell C-indices for assessing the discriminatory ability of biomarkers measured on admission to stratify the risk of 28-day mortality. PESI, Pulmonary Embolism Severity Index; WBC, white blood cell count Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Table 1.Clinical characteristics of patients with acute pulmonary embolism, stratified by 28-day mortality. N= 447 (100 %) 65.0±16.2 193 (43.18) 104.5±37.8 28-day Mortality Survival Death N=401 (89.7 N=46 (10.3 p-value %) %) 64.4±16.4 70.4±13.1 0.016* 171 (42.64) 22 (47.83) 0.502 102.0±37.0 125.9±38.4 <0.001* 123.9±28.6 96.2±21.8 18.4±3.9 94.2±6.5 36.8±0.7 124.9±28.2 95.9±21.3 18.3±3.8 94.3±6.6 36.8±0.7 115.1±30.5 98.6±25.8 19.0±5.1 92.8±6.4 36.9±0.8 0.028* 0.424 0.404 0.132 0.089 55 (12.30) 90 (20.13) 17 (3.80) 47 (11.72) 83 (20.70) 12 (2.99) 8 (17.39) 7 (15.22) 5 (10.87) 0.268 0.380 0.022* 49 (10.96) 63 (14.09) 44 (10.97) 58 (14.46) 5 (10.87) 5 (10.87) 0.983 0.507 32 (7.16) 26 (6.48) 6 (13.04) 0.124 169 (37.81) 42 (9.40) 153 (34.23) 140 (34.91) 38 (9.48) 136 (33.92) 29 (63.04) 4 (8.70) 17 (36.96) <0.001* >0.999 0.681 40 (8.95) 40 (8.95) 8 (1.79) 90 (20.13) 34 (8.48) 39 (9.73) 8 (2.00) 78 (19.45) 6 (13.04) 1 (2.17) 0 (0.00) 12 (26.09) 0.281 0.104 >0.999 0.289 74 (16.55) 65 (16.21) 9 (19.57) 0.562 142 (31.77) 253 (56.60) 372 (83.22) 128 (28.64) 128 (31.92) 233 (58.10) 336 (83.79) 116 (28.93) 14 (30.43) 20 (43.48) 36 (78.26) 12 (26.09) 0.838 0.058 0.342 0.687 63.4±9.9 63.3±10.1 63.7±7.6 0.790 40.7±16.7 168 (37.58) 40.2±16.5 143 (35.66) 45.6±17.4 25 (54.35) 0.069 0.013* 9.9±4.7 7.5±4.4 9.8±4.6 7.3±4.2 12.1±5.5 9.8±5.2 0.001* 0.002* Total Variables Age (years) Male gender [n (%)] PESI score (point) Initial vital sign Systolic blood pressure (mmHg) Heart rate (bpm) Respiratory rate (bpm) O2 saturation (%) Body temperature (°C) Initial symptom [n (%)] Altered mental status Chest pain Hemoptysis Comorbidity [n (%)] Congestive heart failure Coronary artery occlusive disease Chronic obstructive pulmonary disease Malignancy Previous DVT or PE Chronic kidney disease Treatment modality [n (%)] Tissue plasminogen activator Inferior vena cava filter Thrombectomy Usage of LMWH Usage of unfractionated heparin/LMWH Localization of thrombosis [n (%)] Main pulmonary artery Lobar pulmonary artery Segmental pulmonary artery Sub-segmental pulmonary artery Echocardiographic findings Left ventricular ejection fraction (%) RVSP (mmHg) Right ventricular dilatation [n (%)] Laboratory data White blood cell count (10^3/μL) Neutrophil count (10^3/μL) Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Platelet count (10^3/μL) Hemoglobin (g/dL) Creatinine (mg/dL) Creatinine Kinase-MB (mcg/L) Troponin I (mcg/L) D-dimer (mcg/L) hs C-reactive protein (mg/L) Arterial blood PH tCO2 (mmol/L) Delta neutrophil index Time-0 (%) Delta neutrophil index Time-24 (%) 234±124 12.6±2.3 0.9±0.6 3.4±4.1 0.06±0.09 4109±5534 54±69 7.44±0.08 21.2±3.9 1.49±2.94 235±124 12.6±2.3 0.9±0.7 3.4±4.2 0.06±0.09 3745±4292 49±65 7.44±0.07 21.4±3.6 1.16±2.36 2.74±4.78 2.37±4.25 226±126 0.629 11.8±2.2 0.013* 0.9±0.5 0.620 2.7±2.0 0.067 0.05±0.05 0.277 7458±11558 0.045* 102±88 <0.001* 7.43±0.11 0.420 19.7±5.1 0.036* 4.41±5.17 <0.001* 6.20±7.40 0.002* PESI, Pulmonary Embolism Severity Index; DVT, deep vein thrombosis; PE, pulmonary embolism; LMWH, low molecular weight heparin; RVSP, right ventricle systolic pressure; hs C-reactive protein, high sensitivity C-reactive protein. Data are expressed as the mean ± standard deviation or number (percentage). *P<0.05. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Table 2.Multivariable Cox proportional hazard regression analysis for predictors of 28-day mortality in patients with acute pulmonary embolism. Multivariable cox proportional hazard regression analysis (28-day mortality) Variable PESI score (per 1 point) Right ventricular dilatation Hemoptysis on admission White blood cell count (per 10^3/μL) Neutrophil count (per 10^3/μL) Hemoglobin (per 1 g/dL) D-dimer ((per 1 mcg/L)) hs C-reactive protein (per 1 mg/L) tCO2 (per 1 mmol/L) Delta neutrophil index Time-0 (per 1 %) Delta neutrophil index Time-24 (per 1 %) HR (95% CI) p-value HR (95% CI) p-value 1.009 (1.000-1.018) 0.047* 1.007 (0.998-1.016) 0.125 2.618 (1.243-5.513) 0.011* 3.171 (1.450-6.933) 0.004* 4.174 (1.318-13.222) 0.015* 3.124 (0.914-10.683) 0.069 0.869 (0.669-1.130) 0.295 0.783 (0.585-1.048) 0.100 1.158 (0.886-1.515) 0.283 1.315 (0.970-1.783) 0.078 0.956 (0.814-1.124) 0.590 0.923 (0.780-1.091) 0.348 1.006 (1.002-1.011) 0.002* 1.002 (0.996-1.008) 0.570 1.005 (1.001-1.009) 0.015* 1.005 (1.001-1.009) 0.010* 0.961 (0.891-1.036) 0.302 0.945 (0.869-1.028) 0.186 1.107 (1.042-1.177) 0.001* 1.040 (1.001-1.081) 0.047* HR, hazard ratio; CI, confidence interval; PESI, Pulmonary Embolism Severity Index; hs C-reactive protein, high sensitivity C-reactive protein. *P<0.05. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited. Table 3. Sensitivity and specificity analysis of the delta neutrophil index (DNI) to predict 28-day mortality. 28-day mortality (DNI Time-0 >3.0%) Hazard ratio (95% CI) Discovery cohort p-value Internal validation cohort p-value 7.477 (4.183-13.366) <0.001* 7.477 (4.183-13.366) <0.001* 28-day mortality (DNI Time-0 >3.0%) Internal validation Discovery cohort cohort Sensitivity % (95% CI) 54.4 (39.0-69.1) 54.4 (40.0-68.2) Specificity % (95% CI) 88.8 (85.3-91.7) 88.8 (85.4-91.9) Positive predictive value % (95% CI) 35.7 (24.5-46.9) 35.8 (24.7-47.0) Negative predictive value % (95% CI) 94.4 (92.1-96.7) 94.4 (92.0-96.8) Accuracy % (95% CI) 85.2 (81.9-88.5) 85.3 (81.9-88.6) DNI, delta neutrophil index; CI, confidence interval. *P<0.05. The optimal cut-off point was determined in the discovery cohort by using Contal and O’Quigley’s method. A cut-off of 3% was applied to the population, and the sensitivity and specificity was estimated in DNI. When the same cut-offs were applied to the validation cohort using bootstrapping (mean HR of 1,000 repetitions), the Sensitivity and specificity analysis obtained by internal validation were similar to deviation cohort. Copyright © 2017 by the Shock Society. Unauthorized reproduction of this article is prohibited.