Study population
This study was performed in the 30-bed medical ICU of Severance Hospital in Seoul, Korea. The protocol was approved by the Institutional Review Board, and written informed consent was obtained from the patients or next of kin.
Over a period of 6 months (from July 2010 to December 2010), all consecutive patients with clinically diagnosed sepsis at the time of ICU admission were included. Systemic inflammatory response syndrome (SIRS) was defined as two or more of the following conditions: (a) body temperature > 38°C or < 36°C; (b) leukocytosis (> 10,000/μl), leukopenia (< 4,000/μl), or > 10% bands; (c) heart rate > 90 beats/min; and (d) respiratory rate > 24 breaths/min. Sepsis was defined as SIRS with proven or suspected microbial etiology. Severe sepsis was defined as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion. Septic shock was defined as an acute circulatory failure characterized by persistent arterial hypotension (systolic arterial pressure below 90 mmHg, mean arterial pressure < 60 mmHg, or a reduction in systolic pressure of > 40 mmHg from baseline despite adequate fluid resuscitation in the absence of other causes of hypotension) [19]. Exclusion criteria were age less than 18 years, pregnancy, patients with hematologic abnormalities, and those who received granulocyte colony stimulating factors, glucocorticoid, or other immunosuppressants before study enrollment.
Patients originated either from the emergency room or from the general wards. At the time of notification for ICU admission of each patient, every effort was made to identify patients with suspected sepsis by dedicated research fellows and attending physicians. Microbiological tests were performed on blood samples, sputum (by nasopharyngeal swab or endotracheal suction), urine specimens, removed catheters, and secretions from other body regions which were suspected to be the infection source. All patients underwent chest radiography and/or high resolution computed tomography scan, magnetic resonance imaging scan, or endoscopy when indicated by attending physicians without interference by the study investigators. On the basis of laboratory, bacteriological, and radiographic findings, "confirmed infection" was determined by a definite source of infection (microbiology confirmed and/or positive culture at a likely focus), and "probable infection" was determined by positive imaging findings, such as an infiltration, cavity, or abscess confirmed by specialized radiologists. Thus, clinically diagnosed sepsis was defined by either confirmed infection or probable infection under the presence of SIRS. During ICU stay, all patients were treated following the international guidelines for management of severe sepsis and septic shock [20].
Data collection
Baseline demographic data and clinical variables, including age, sex, blood pressure, pulse rate, primary site of infection, blood culture results, presence of severe sepsis/septic shock or overt DIC, average amount of norepinephrine infusion, urine output during the first 24 hours after ICU admission, and hospital mortality were recorded. The presence or absence of mechanical ventilation and renal replacement therapy were also recorded. Simplified Acute Physiology Score (SAPS) 3 [21, 22] and Sequential Organ Failure Assessment (SOFA) score [23] were calculated on ICU admission to measure the severity of patient condition. Overt DIC was defined as DIC score ≥ 5 based on the diagnostic criteria by the International Society of Thrombosis and Hemostasis [24].
DNI and other blood sample measurements
Blood samples for the analyses of DNI and other laboratory parameters were obtained from indwelling arterial catheters or by venipuncture within the first 24 hours of ICU admission. The blood samples were drawn from each patient into EDTA tube, and were immediately transported at room temperature to the chemical laboratory department, and the assay was performed within 1 hour of blood sampling.
A specific type of automatic cell analyzer (ADVIA 2120 Hematology System, Siemens Healthcare Diagnostics, Forchheim, Germany) was used for calculating DNI. This is a flow cytometry-based hematologic analyzer which has two independent white blood cell (WBC) analysis methods, an MPO channel and lobularity/nuclear density channel. First, after lysis of red blood cells (RBCs), the tungsten-halogen based optical system of the MPO channel measures cell size by forward light scatter, and stain intensity by absorbance, thereby counting and differentiating granulocytes, lymphocytes, and monocytes based on their size and MPO content. Second, the laser diode-based optical system of the lobularity/nuclear density channel counts and classifies cells according to size, lobularity, and nuclear density [17, 18]. The formula for calculating DNI is as follows: DNI = [the neutrophil subfraction and the eosinophil subfraction measured in the MPO channel by cytochemical MPO reaction] - [the PMN subfraction measured in the nuclear lobularity channel by the reflected light beam]. The correlation between DNI values and immature granulocytes by manual counting was reported in a previous study [18]. The measurement of immature granulocytes included promyelocytes, myelocyte, and metamyelocytes, but not blasts.
Complete blood cell counts, including WBC count and absolute neutrophil count (ANC), were measured with an automated analyzer (ADVIA 2120 Hematology System). Prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer, and fibrinogen levels were assayed using an STA analyzer (Diagnostica Stago, Asnieres-Sur-Seine, France). Antithrombin III activity was determined using an ELISA kit (Diagnostica Stago). Plasma C-reactive protein (CRP) concentration was measured by direct immunoturbidimetry (CA400, Beckman Coulter, CA, USA). Lactate levels were measured in arterial blood using point-of-care blood gas analyzers (Critical Care Xpress, NOVA biomedical, MA, USA). All measurements were performed according to the manufacturers' instructions.
Statistical analysis
Continuous variables are presented as the mean ± standard deviation (SD), or when the assumption of normality was violated, as median values and interquartile range. Categorical variables were expressed as absolute and relative frequencies. Comparisons between groups were performed with chi-squared tests for categorical variables and Mann-Whitney U test or Kruskal-Wallis test for continuous variables, as appropriate. We classified patients according to the severity of sepsis (sepsis, severe sepsis, and septic shock), and compared the values of DNI and other laboratory biomarkers among the groups. If statistically significant, post-hoc analysis was performed using the Dunn procedure. For comparison, we presented the value of DNI in healthy subjects as controls. The correlation between DNI and other laboratory variables or clinical severity scores was tested by Spearman's method. The effect of increasing quartiles of DNI on the proportion of overt DIC or severe sepsis/septic shock was evaluated by the Cochrane-Armitage trend test. Receiver-operating characteristics (ROC) curves were constructed and the Youden Index method was used to determine the optimal cut-off values for DNI, WBC, ANC, lactate, and CRP for predicting severe sepsis/septic shock. The areas under the curves (AUCs) were calculated to compare the diagnostic performance of each marker. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using SAS software, version 9.2 (SAS Institute Incorporated, Cary, NC, USA).