B-075 Biophysical Changes of Leukocyte Activation (and NETosis) in the Cellular Host Response to Sepsis

Abstract

Abstract Background During sepsis, neutrophils, upon activation, undergo biophysical and biochemical changes that may terminate in the release of neutrophil extracellular traps (NETs) (1–3). The IntelliSep test is a rapid sepsis diagnostic that assesses immune activation by quantifying biophysical properties of leukocytes from whole blood in <10 minutes. The test results in the IntelliSep Index (ISI), ranging between 0.1–10.0, stratified into three interpretation bands (Band 1, Band 2, and Band 3) based on the probability of the clinical syndrome of sepsis (4, 5). We investigated the correlation between the IntelliSep result and immunological changes observed during leukocyte activation through in-vitro studies and using clinical samples from non-septic and septic patients. Methods In-vitro Studies: Healthy blood samples were collected between April - August 2022 from 18 volunteers at a blood donor center. Aliquots of each blood sample were incubated for 10 minutes with phorbol myristate acetate (PMA) in phosphate-buffered saline at one of 3 concentrations: 0, 200, and 400 nM prior to the IntelliSep test (3 repeats per donor per concentration). Clinical Studies: Subjects with signs or symptoms of infection were enrolled from Emergency Departments (EDs) in three similar but distinct prospective cohort studies (February 2016—September 2019) (1, 2). The IntelliSep test was performed on a aliquot of fresh whole blood from each subject, and remnant plasma was prepared and frozen for later analysis. Plasma levels of neutrophil elastase-DNA (NE DNA) and citrullinated histone H3-DNA (Cit-H3 DNA) complexes were quantified using previously reported custom ELISAs 3,6. Subjects were retrospectively adjudicated as sick (adjudicated as septic with SOFA scores peaking on the day of enrollment compared to two subsequent days) and “healthy” (SOFA for day of enrollment <2, hospital length of stay <3 days). Results Significant increases in ISI scores were observed with increasing concentration of PMA in healthy blood samples (0 and 200: P < 10−10; 0 and 400: P < 10−10). The clinical analysis cohort consisted of 39 “sick” and 42 “healthy” subjects. Linear correlation was observed between the ISI and NE DNA and Cit-H3 DNA quantities with significant increases across ISI Interpretation Bands (Band 1 to Band 3: P < 0.001). Conclusion Correlation of increasing ISI and PMA concentration supports the hypothesis that the biophysical changes measured by the IntelliSep test result from leukocyte activation. Observed correlation between the ISI and NET quantities suggest that NET formation is one of the activation pathways that result in the biophysical changes measured in the ISI. Together these experiments support that biophysical changes of leukocyte activation measured using a cellular host response test could provide a window into a patient’s state of dysregulated immunity and may have the potential to aid ED physicians in timely diagnosis of sepsis.