Diagnoses and the number of patients exhibiting neutrophil extracellular traps and citrullinated histone H3 in each diagnostic group.

<p>In the blood smears surveyed in this study, we identified NETs in 5 patients and Cit-H3 in 11 patients. Both NETs and Cit-H3 were identified concurrently in one patient with infection. We found no NETs or Cit-H3-positive cells in samples from patients with trauma (0/7) or heart disease (0/4). NETs: neutrophil extracellular trap, Cit-H3: citrullinated histone H3.</p><p>Diagnoses and the number of patients exhibiting neutrophil extracellular traps and citrullinated histone H3 in each diagnostic group.</p

Results of single logistic regression analysis.

<p>Logistic regression analysis was performed to identify the factors related to the presence of NET and Cit-H3 in the bloodstream. Only “the presence of bacteria in tracheal aspirate” (+) at the time of intubation was a significant factor associated with the presence of NET and Cit-H3 (<i>p</i> = .0112). NETs: neutrophil extracellular traps, Cit-H3: citrullinated histone H3, SIRS: systemic inflammatory response syndrome, cf-DNA: circulating free DNA, WBC: white blood cell, IL: interleukin, TNF: tumor necrosis factor, HMGB1: high mobility group box-1.</p><p>Results of single logistic regression analysis.</p

Results of multiple logistic regression analysis of factors associated with the presence of neutrophil extracellular traps and/or citrullinated histone H3.

<p>Two methods of multiple regression analysis, backward and forward regression, yielded similar models. “The presence of bacteria in tracheal aspirate” was the only factor that was significantly related to the presence of neutrophil extracellular traps and/or citrullinated histone H3 in the bloodstream. The odds ratio for aspiration was 5.750. Coeff (β): coefficient; OR: odds ratio, Lower: lower level of 95% confidence interval, Upper: upper level of 95% confidence interval.</p><p>Results of multiple logistic regression analysis of factors associated with the presence of neutrophil extracellular traps and/or citrullinated histone H3.</p

Representative images of immunostaining using anti-CD66b antibody in the blood smear sample from a critically ill patient.

<p>Triple staining by DAPI, anti-CD66b antibody, and anti-citrullinated histone H3 was performed using the blood smear sample obtained from a critically ill patient. A. The CD66b-positive cells were subjected to citrullination of histone H3 in their nuclei. Citrullination of histone H3 was not detected in the CD66b-negative cell (arrow). B. Arrow indicates the occurrence of citrullination of histone H3 in a neutrophil that had immunoreactivity against CD66b. Arrowheads indicate NETs stained with CD66b, whose appearance was of a string-like structure extending from the cell body. Asterisk indicates a neutrophil that was beginning to release NETs from its ruptured cell body. Interestingly, freshly produced NETs (asterisk) held immunoreactivity against citrullination of histone H3. In contrast, elongated NETs (arrowheads) were not stained with anti- citrullinated histone H3 antibody. Blue, DAPI; Red, CD66b; Green, citrullinated histone H3. (Magnification ×400). Scale bar; 50 µm.</p

Comparison between patients positive and negative for neutrophil extracellular traps and/or citrullinated histone H3.

<p>Among the factors evaluated to highlight the relation to the presence of NETs or Cit-H3 in the bloodstream, only “the presence of bacteria in tracheal aspirate” differed significantly between the NET- and/or Cit-H3-positive and -negative groups (<i>p</i><.01). The other factors were not significantly related to the presence of NETs and/or Cit-H3. Continuous variables are presented as the median and IQR unless otherwise noted. The Wilcoxon rank-sum test and Pearson's chi-square test were used to compare two patient groups. NETs: neutrophil extracellular traps, Cit-H3: citrullinated histone H3, IQR: interquartile range, APACHE: Acute Physiological And Chronic Health Evaluation, SOFA: Sequential Organ Failure Assessment, SIRS: systemic inflammatory response syndrome, WBC: white blood cell, IL: interleukin, TNF: tumor necrosis factor, cf-DNA: circulating free DNA, HMGB1: high mobility group box-1.</p><p>Comparison between patients positive and negative for neutrophil extracellular traps and/or citrullinated histone H3.</p

Patient characteristics.

<p>During the study period, 263 patients were admitted to the ICU of whom 49 were intubated and were included in this study. We excluded patients with cardiopulmonary arrest who could not be resuscitated on admission. IQR: interquartile range, APACHE: Acute Physiological And Chronic Health Evaluation, SOFA: Sequential Organ Failure Assessment, SIRS: systemic inflammatory response syndrome, WBC: white blood cell.</p><p>Patient characteristics.</p

Representative images of immunofluorescence staining to detect neutrophil extracellular traps (NETs).

<p>NETs were visualized in the blood smear samples by immunocytochemistry and identified as extracellular string-like structures composed of chromatin (DNA and histone H3). NETs were present in the bloodstream of critically ill patients. Citrullination of histone H3 was not recognized in these images. In the blood smears surveyed in this study, we identified NETs in five patients (5/49, 10.2%). Blue, 4′,6-diamidino-2-phenylindole (DAPI); red, histone H3; green, citrullinated histone H3. Arrowheads indicate the double-stained areas containing NETs (Magnification ×400). Scale bar; 50 µm.</p

Representative images of immunofluorescence staining to detect citrullinated histone H3 (Cit-H3).

<p>Citrullination of histone H3, which is a critical enzymatic process to produce NETs through decondensation of chromatin, was visualized in the blood smear samples using anti-citrullinated histone H3 antibody by immunohistochemistry. Cit-H3 was present in the bloodstream of critically ill patients. The inset in the merged image is the magnified image of a representative cell (white rectangle) expressing citrullinated histone H3 in the nucleus. Neutrophil extracellular traps are not recognized here. In the blood smears surveyed in this study, we identified Cit-H3 in 11 patients (11/49, 22.4%). Blue, 4′,6-diamidino-2-phenylindole (DAPI); red, histone H3; green, citrullinated histone H3 (Magnification ×400). Scale bar; 50 µm.</p