Study design
The Hellenic Sepsis Study Group has been collecting the data of patients with sepsis since 2006 from 55 study sites across Greece (20 intensive care units and 35 emergency departments (EDs) and departments of internal medicine or surgery). The patients were enrolled after written consent was provided by themselves or by their first degree relatives (for patients unable to consent). The protocol was submitted and accepted from the ethics committees of the participating hospitals. The study involved patients with suspected infection plus at least two SIRS criteria, i.e., sepsis according to the original sepsis definition [
2]. Patients infected with the human immunodeficiency virus (HIV) and patients with neutropenia were excluded. Infections and organ dysfunction were defined according to already-published international criteria [
10]. From all patients 10 ml blood was collected after peripheral venipuncture within the first 24 h of SIRS presentation, and the procedure was repeated after 48 h. Serum was prepared by centrifugation at 900 g, and samples were transported within the same day to the central lab and stored at – 80 °C until processing.
Clinical data were collected: comorbidities, the Charlson’s comorbidity index, gender, age, medical history, vital signs, complete physical examination including assessment of Glasgow Coma Scale, type of infection, absolute blood cell count, international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, fibrinogen degradation products, glucose, urea, creatinine, Na+, K+, albumin, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, bilirubin, arterial pH, partial oxygen pressure, partial carbon dioxide pressure, bicarbonate, lactate, and ratio of partial oxygen pressure to fraction of inspired oxygen (pO2/FiO2). Blood cultures from peripheral veins and central lines were performed as well as urinalysis, quantitative urine cultures, and quantitative cultures of tracheobronchial secretions, if necessary. Chest X-ray, abdominal ultrasound, and chest and abdominal computed tomography were also performed, if necessary. Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores as well as data about 28-day outcome were collected.
Commercial enzyme immunosorbent assays were used for the measurements of ferritin (ORGENTEC Diagnostika GmbH, Mainz, Germany), sCD163 (Affymetrix Inc., Santa Clara, CA, USA), tumor necrosis factor (TNF) alpha (R&D Systems, Inc., Minneapolis, MN, USA), IL-6 (Affymetrix Inc.), IL-10 (R&D Systems, Inc.), IL-18 (OriGene Technologies Inc., Rockville, MD, USA), and interferon gamma (IFN-γ) (Affymetrix Inc.). The lower detection limit of ferritin was 5 ng/ml, of sCD163 0.31 ng/ml, and of all cytokines 20 pg/ml. Triglycerides were measured in serum with Lipase/GPO-Trinder (Siemens Healthcare Diagnostics Inc.), and the lower detection limit was 8 mg/dl. All measurements were performed in duplicate and reported by technicians blind to clinical information.
The study endpoints were (1) the frequency of MALS in septic patients using predefined criteria and (2) the development of a biomarker for the early recognition of MALS. For this purpose, the patients were divided into two cohorts, a test cohort and a validation cohort. Patients were randomized into a test cohort and a validation cohort depending on study site and enrollment date in a 2:1 ratio.
Criteria for MALS and Sepsis-3
The criteria that we used for the classification of MALS were not the same as those suggested by other authors. However, we developed a classification system that could provide equivalent criteria for classification. This is the reason why we classified patients as having MALS and not MAS. More precisely, MALS was diagnosed in every patient who either met the HScore 2014 suggested for adults suffering from autoimmune diseases [
15] and/or had both DIC and HBD, as suggested by Shakoory et al. [
5]. The HScore, suggested for the diagnosis of hemophagocytosis syndrome (HS), provides specific points for the following variables: immunodeficiency (defined as infection by HIV and/or long-term treatment with immunosuppressive drugs such as cyclosporine, glucocorticoids, and azathioprine; up to 18 points), body temperature (up to 49 points), organomegaly (up to 38 points), cytopenias (up to 34 points), serum ferritin (up to 50 points), triglycerides (up to 64 points), fibrinogen (up to 30 points), aspartate aminotransferase (up to 19 points), and hemophagocytosis in the bone marrow (up to 35 points). The final score varies between 0 and 337 points, so that scores higher than 169 are associated with 90% sensitivity for HS. In the current study, the HScore could range between 0 and 302, since bone marrow aspiration providing 35 points (i.e., 10.4% of the maximal points) was not done routinely in our patients. Taking into consideration that in the original HScore the cutoff value of 169 represented the median of the HScore, it seemed logical to use the median of 151 as the new cutoff value. As a consequence, this modification comprising eight criteria could not be the same as the original HScore, which uses nine criteria. However, it was anticipated to be equivalent to the original HScore. Moreover, we tried to adjust for this limitation in a conservative approach: patients presenting with both HBD and DIC as suggested by Shakoory et al. [
5] were also classified as having MALS. HBD was defined by the presence of at least two of the following: (1) serum bilirubin higher than 2.5 mg/dl, (2) aspartate aminotransferase at least two times higher than the upper normal limit, and (3) INR higher than 1.5. DIC was not defined using the definition applied in the manuscript by Shakoory et al. [
5]. Instead, in order to achieve better diagnostic sensitivity for DIC, the DIC Score of the International Society of Thrombosis and Hemostasis (ISTH) was used, providing points for the absolute platelet count (maximum 2 points), elevated fibrin-related markers (maximum 3 points), prolonged prothrombin time (maximum 2 points), and fibrinogen level (maximum 1 point). Patients with scores more than or equal to 5 were considered to have overt DIC [
16]. All enrolled patients were reclassified into infection and sepsis categories using the new Sepsis-3 definition [
17]. Those classified as having sepsis by the new classification and who scored positive for HS and/or HBD/DIC were diagnosed with MALS.