Neutrophil migration parameters in burn patients.

<p>(A) Heat-map showing the results of 18 neutrophil migratory phenotype measurements in 74 blood samples from 13 burn patients and three healthy volunteers. Specific measurements are explained in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114509#pone-0114509-t002" target="_blank">Table 2</a>, including averages for each parameter. Each parameter was measured in three conditions: in the absence of chemoattractant, and in the presence of fMLP and LTB₄ chemoattractant gradients. Bright green illustrates 2 S.D. below healthy donor averages, light green illustrates 1 S.D. below healthy donor averages, bright red illustrates 2 S.D. above healthy donor averages, and light red illustrates 1 S.D. above healthy donor averages. Below the heat map, a color coded bar represents the status of the blood donor at the time of the draw. Red illustrates sepsis, green illustrates SIRS, and black illustrates no SIRS status. The day post burn is indicated above the bar, the patient identifier is presented below the bar. Healthy donors, burn patients with sepsis, and burn patients without sepsis are grouped by the accolades. (B) Kymograph showing an example of oscillatory migration of a neutrophil from a septic burn-patient, in the absence of chemoattractant. The time interval between successive frames is 2 minutes. Vertical scale bar is 100 µm.</p

Correlations between the changes in neutrophil spontaneous migration score (NAS_N) and the timing of sepsis in burn patients.

<p>(A) Neutrophil activation score averages are significantly different between burn patients with sepsis (N = 21) and patients with SIRS (N = 29, P = 0.038) or patients with no SIRS (N = 21, P = 0.027). (B) The NAS_N is significantly higher in patients in samples before clinical diagnosis (P = 0.036) and drops to one third following septic diagnosis and post-antibiotic therapy. (C) ROC curves for averaged NAS_N of two successive blood samples demonstrates the good predictive values of the spontaneous neutrophil migration patterns for sepsis in patients with major burns.</p

The components and temporal evolution of neutrophil spontaneous migration score (NAS_N) in patients with major burns.

<p>(A) Two dimensional comparisons between neutrophil migration parameters between healthy donors (black stars), burn patients with no complications (green square), SIRS (green triangle), and sepsis (red square). In the absence of chemoattractant, neutrophils from patients with sepsis migrate in larger numbers and display more oscillatory migration than the patients with SIRS or those from healthy donors (which do not migrate). (B) Neutrophils from patients with sepsis migrate longer distances and display more oscillatory migration than the patients with SIRS or those from healthy donors (which do not migrate). (C) Changes in the neutrophil activation score in the 6 patients with no sepsis during the hospital stay. On patient that died is indicated (red cross). (D) Changes in the neutrophil activation score in the 7 patients that experience sepsis during the hospital stay. Higher scores are observed during sepsis (filled red triangle). In patients that respond to antibiotic treatment, NAS_N decreases when sepsis is resolved (empty red triangles). In some of the patients, NAS_N increased even several days before sepsis was diagnosed (red star). The NAS_N remained low in these patients in the absence of complications (empty gray triangle).</p

Measuring neutrophil migration in burn patients during sepsis.

<p>(A) Overview of the protocol using microfluidic devices to measure neutrophil migration in patients with major burns. (1) Three milliliters blood samples are obtained from patients with major burns. (2) Neutrophils are separated from blood using negative selection beads. (3) Microfluidic devices are primed with chemoattractant solutions and patient neutrophils are introduced into the neutrophil loading channel. (4) Neutrophils are observed using time-lapse imaging during migration through 3×6 µm channels and mazes of channels. (5) Images of moving neutrophils are recorded at single cells resolution and analyzed to quantify 18 neutrophil migratory parameters. (6) A neutrophil spontaneous motility score is calculated for each sample. (7) Correlations are tested between the Neutrophil activation score (NAS_N) and sepsis in patients with major burns. This cycle is repeated every two days or less often for the duration of burn patient treatment, up to one month. (B) Typical timeline for sample collection from burn patients. During the first phase, samples are collected every two days for one week. A second phase continues with one sample every week for up to one month. A third phase is triggered by the clinicians when there is request for blood cultures, signs of fever, leukocytosis and/or thrombocytosis. During Phase three, samples are collected every two days for a week.</p

Summary of burn patient enrollment and hospitalization statistics.

<p>Summary of burn patient enrollment and hospitalization statistics.</p

Summary of neutrophil phenotypic migration parameters to an array of chemoattractants (no chemoattractant, fMLP, and LTB₄).

<p>Averages +/− standard deviations are shown for all parameters for both healthy donors and burn patients. • indicates average parameters that are significantly different between healthy donors and burn patients, (p<0.05)</p><p>Summary of neutrophil phenotypic migration parameters to an array of chemoattractants (no chemoattractant, fMLP, and LTB₄).</p