Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity

Abstract

Dysregulated leukocyte responses underlie the pathobiology of sepsis, which is a leading cause of death. However, measures of leukocyte function are not routinely available in clinical care. Here we report the development and testing of an inertial microfluidic system for the label-free isolation and downstream functional assessment of leukocytes from 50 μl of peripheral blood. We used the system to assess leukocyte phenotype and function in serial samples from 18 hospitalized patients with sepsis and 10 healthy subjects. The sepsis samples had significantly higher levels of CD16 dim and CD16 − neutrophils and CD16 + 'intermediate' monocytes, as well as significantly lower levels of neutrophil-elastase release, O 2 − production and phagolysosome formation. Repeated sampling of sepsis patients over 7 days showed that leukocyte activation (measured by isodielectric separation) and leukocyte phenotype and function were significantly more predictive of the clinical course than complete-blood-count parameters. We conclude that the serial assessment of leukocyte function in microlitre blood volumes is feasible and that it provides significantly more prognostic information than leukocyte counting.

Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics

“…In addition, granulocytes were isolated from peripheral blood collected from healthy donors after informed consent was obtained, using the density-based gradient over Histopaque 1077 (Sigma-Aldrich) and Histopaque 1119 (Sigma-Aldrich) as previously described. 1,35…”

“…PMN phagolysosome formation was quantified through flow cytometry using the red E. coli pHrodo bioparticle conjugate as described in our previous work. 1…”

“…[1][2][3][4][5][6][7][8] Since erythrocytes or red blood cells (RBCs) are the most abundant cell component (approximately 1000 times more than the number of WBCs) in peripheral blood, for effective and reliable leukocyte assessment, the depletion of RBCs is an essential sample preparation step before any kinds of scientific, clinical, and diagnostic tests of WBCs. 1,4,6,9 As the current standard for blood cell separation in the laboratory, density gradient centrifugation is the most widely used technique. Other affinity-based (labeling) methods, such as fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS), have been used for precise control and separation of target cells.…”

“…Other affinity-based (labeling) methods, such as fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS), have been used for precise control and separation of target cells. 1,[9][10][11][12][13] Although those methods themselves are straightforward, they entail many time-consuming steps and require well-trained technicians and well-equipped laboratories; for example, approximately 2-3 hours are needed for a typical density gradient centrifugation. [9][10][11] In addition, for their proper operation, a large volume of blood is required (usually 30-45 mL of blood is required for density gradient centrifugation), and expensive labeling reagents should be employed for the labeling methods.…”

“…More importantly, the centrifugation process and cell labeling can cause ex vivo cell activation, especially for sensitive cell types such as neutrophils and macrophages, which poses challenges to the correct assessment of the host immune response or leukocyte functions. 1,4,14,15 Generally, continuous blood assessment is required for monitoring the host immune response of patients, which can cause iatrogenic anemia (anemia caused by repeated blood drawing) in critically ill patients. 1,16 In the next generation of sample preparation, for more precise and effective analysis, daily or even hourly sample assessment should be processed in the bedside or in a field-deployable manner, which requires compact and portable equipment carrying out easy (without well-trained personnel), fully-automated (or even hand-operable), reliable, and fast (within 10 minutes) operation using microliter sampling without any sample damage.…”