Assaying leukocyte hallmarks during sepsis

Understanding disease pathology, monitoring disease progression and evaluating treatment efficacy often involve the measurement of immune responses to the disease. Across disease types, progress in the development of technologies for quantifying immune responses has been uneven. For example, immune responses against cancer are reasonably understood (which has helped fuel fast progress in cancer immunotherapies). By comparison, knowledge of the deterioration of immune responses during sepsis (which has a mortality rate of about 25%) remains rather primitive. The probing of acquired immunity (both antibody and cell-mediated responses) via the analysis of B lymphocytes and T lymphocytes by flow cytometry is well-established, both in academic research and in clinical laboratories, yet functionally analysing cells of the innate immune system (in particular, neutrophils and monocytes) remains mostly a research endeavour. This is partly because of activation artefacts arising from procedures for leukocyte isolation from blood, and because the current assays are labour-intensive. Reporting in Nature Biomedical Engineering, Bruce Levy and colleagues now show that a set of functional measurements of leukocytes, made by processing just 50 μL of peripheral blood from sepsis patients via an array of microfluidic assays, can be performed faster and more precisely, and that the measurements correlate with the clinical status of the patients after they have been diagnosed¹.

Levy and co-authors’ microfluidic setup used inertial focusing² of whole-blood samples to separate neutrophils and monocytes. Downstream of the separation device, conventional flow-cytometry techniques available in clinical laboratories probed the neutrophils and monocytes for the expression of surface markers, phagocytosis and acidification of phagosomes (the vesicles engulfing the phagocytized material), the production of reactive oxygen after the stimulation of the cells, and the production of the proteolytic enzyme elastase after cell activation.

By using isodielectric-point measurements in microfluidic channels, the authors also assessed the release of neutrophil extracellular traps (extracellular fibrous networks that bind pathogens). Then, they leveraged the three technologies (inertial focusing, flow cytometry and isodielectric separation), which they optimized to be able to use blood volumes of only tens of microliters, to comprehensively characterize the neutrophils and monocytes of 18 sepsis patients repeatedly for one week after sepsis diagnosis. Compared with healthy individuals, the functions of the neutrophils from the sepsis patients were already significantly altered in the first day after diagnosis, and correlated with sepsis severity on patient admission. Neutrophil expression of CD16, monocyte expression of CD16 and CD14, neutrophil elastase release and reactive-oxygen production all recovered partially seven days after diagnosis, independently of patient outcome. Changes in surface markers were overall consistent with previously reported differences in neutrophil functions measured on cells purified via traditional density-gradient-based protocols, and in patients that develop sepsis after severe trauma³ and after major burn injuries⁴. The changes in elastase release and reactive-oxygen production also appear to be consistent with those determined for other neutrophil functions (in particular, chemotaxis and spontaneous motility) in sepsis patients⁵. Notably, the phenotypic and functional biomarkers tracked the patients’ clinical course more accurately than complete blood counts and clinical measures of sepsis severity (such as the sequential organ-failure assessment score; Fig. 1).

Fig. 1|. Performance of different classes of sepsis biomarkers.

a-d, Loading plots (obtained via multivariate principal-component analyses) for routine measures of complete blood count (a); aggregate indicators of clinical severity (including the need for vasopressors and mechanical ventilation), and the APACHE II (acute physiology, age, chronic health evaluation II) and SOFA (sequential organ failure assessment) scores (b); biomarkers of leukocyte phenotype (c); and functional responses of neutrophils (d) in 11 sequential samples from five sepsis patients, and in nine healthy patients. Only the aggregate phenotypic and functional measurements of leukocytes (as indicated by the reduced overlap of the coloured areas in c and d) distinguished sepsis patients from healthy ones, as well as sepsis patients by day after diagnosis. Figure reproduced from ref. ¹, Springer Nature Ltd.

For microfluidic assays of neutrophil and monocyte function to be useful at the bedside, the promising correlations between the functional alterations of the leukocytes and clinical severity would need to be tested in studies enrolling large numbers of patients. Large and diverse patient cohorts may also help determine whether the assays can accurately distinguish patients in the early stages of sepsis and predict patient outcomes in response to treatments. Also, the monitoring of patients in critical conditions who do not develop sepsis may help determine thresholds between beneficial and detrimental functional changes in cells of the innate immune system. Moreover, the integration of functional changes of leukocytes with changes of gene expression in these cells during sepsis⁶, with additional biomarkers (such as CD64, which is found in monocytes; ref. ⁷) and with comprehensive analyses of cytokines and of other biomarkers⁸, may help find better (and even personalized) biomarkers of sepsis that integrate information at the molecular and cellular scales.

Voltage changes during cell depolarization in the heart, which are recorded by electrocardiograms, have long provided physicians with quantitative metrics that are directly relevant to the detection of cardiac pathologies. For the much less studied pathologies caused by a dysregulated immune system, Levy and colleagues’ quantitative microfluidic assays for the functional analysis of innate immune cells may spur the development of new diagnostic assays and of quantitative diagnostic and prognostic biomarkers.