Assessing neutrophil-derived ROS production at the bedside: a potential prognostic tool in severe COVID-19 cases.

PURPOSE: A prompt and effective immune response is required for clearance of pathogens but exaggerated states of inflammation can cause extensive collateral damage to the host. We have previously used a rapid near-patient assay that measures the functional capacity of neutrophils to produce reactive oxygen species (ROS) to show that values are elevated in patients with severe COVID-19 or sepsis. Here, we assess the utility of longitudinal ROS measurements to monitor and predict mortality outcome for patients with COVID-19 infection being treated in an ICU setting. METHODS: We used the Leukocyte ImmunoTest™ (LIT™) to quantify neutrophil ROS release using a small volume (10 µL) of capillary blood in a portable, rapid (10-min) format. RESULTS: ROS values (LIT score) and ROS levels assessed in relation to neutrophil count (LIT/N) were both markedly elevated in the patient group. Furthermore, these correlated strongly with peripheral neutrophil count and CRP value. Serial measurement of neutrophil or CRP values were not able to reliably predict mortality within the study. In contrast, LIT and LIT/N values started to decline at 7 and 5 days, respectively, in patients who survived ICU admission and this increment increased further thereafter. CONCLUSIONS: This study raises the possibility of LIT and LIT/N to be used as a predictive clinical tool for patients with severe COVID-19 and argues for its assessment to inform on prognosis, and potentially guide treatment pathways, in other disorders associated with neutrophil activation. TAKE-HOME MESSAGE: A longitudinal study of 44 severe COVID-19 patients in the ICU of a leading teaching hospital has demonstrated the prognostic potential of a rapid bedside assay of neutrophil-derived reactive oxygen species (ROS). Assessment of changes in ROS production, as measured using the Leukocyte ImmunoTest™, shows that ROS production generally declined back to normal levels for patients who survived, but remained elevated for those patients who did not survive.

High generation of reactive oxygen species from neutrophils in patients with severe COVID-19.

Neutrophilia and an elevated neutrophil:lymphocyte ratio are both characteristic features of severe COVID-19 infection. However, functional neutrophil responses have been poorly investigated in this setting. We utilised a novel PMA-based stimulation assay to determine neutrophil-derived reactive oxygen species (ROS) generation in patients with severe COVID-19 infection, non-COVID related sepsis and healthy study participants. ROS production was markedly elevated in COVID-19 patients with median values ninefold higher than in healthy controls and was particularly high in patients on mechanical ventilation. ROS generation correlated strongly with neutrophil count and elevated levels were also seen in patients with non-COVID related sepsis. Relative values, adjusted for neutrophil count, were high in both groups but extreme low or high values were seen in two patients who died shortly after testing, potentially indicating a predictive value for neutrophil function. Our results show that the high levels of neutrophils observed in patients with COVID-19 and sepsis exhibit functional capacity for ROS generation. This may contribute to the clinical features of acute disease and represents a potential novel target for therapeutic intervention.

Quantifying transient psychological stress using a novel technique: changes to PMA-induced leukocyte production of ROS in vitro.

Although much work has been conducted to quantify the long-term physiological effects of psychological stress, measures of short-term, low-level stress have been more elusive. This study assessed the effect of exposure of volunteers to a mild, brief, psychologically stressful event, on the functional ability of leukocytes in whole blood to respond to phorbol 12-myristate 13-acetate (PMA) in vitro. Volunteers operated a car electric window and adjusted it to 4 pre-determined positions. Between each operation the mechanism's polarity was covertly altered (group B) or remained unaltered (group A). For each treatment group 10 different subjects provided capillary blood samples pre- and post-stressor. Using a chemiluminescent technique termed leukocyte coping capacity, the ability of leukocytes to produce reactive oxygen species (ROS) in vitro was assessed. ROS release differed significantly at 10 min post-stressor between treatment groups, suggesting exposure to acute psychological stress leads to a reduced ability to respond to bacterial challenge.

Leukocyte counts and neutrophil activity during 4 h of hypocapnic hypoxia equivalent to 4000 m.

INTRODUCTION: Symptoms and signs of infectious disease are increased in subjects at altitude. Most infections at altitude are diagnosed clinically and do not have objective data to support the diagnosis. Since in vivo innate immune responses to hypoxia have not been thoroughly characterized, we investigated the effect of acute systemic hypocapnic hypoxia on leukocyte trafficking and neutrophil activity in healthy humans at rest. METHODS: Sixteen male subjects [mean +/- SD age 28.3 +/- 6.5 yrs, body mass 80.9 +/- 15.9 kg, Vo2peak 4.10 +/- 0.76 L x min(-1)] breathed a hypoxic gas mixture (F(IO)2 = 12.2%, equivalent to 4000 m; H) or normoxic room air (F(IO)2 = 20.9%; N) for 240 min, via a mouthpiece, followed by 60 min of normal breathing. RESULTS: H induced a differential response in peripheral venous blood neutrophils (p < 0.05), lymphocytes (p < 0.01), and eosinophils (p < 0.01; 60-240 min), resulting in a relative lymphopenia (H 1.88 +/- 0.48 and N 2.14 +/- 0.45 x 10(9) L(-1)) and neutrophilia (H 5.2 +/- 1.8 and N 3.9 +/- 1.1 x 10(9) L(-1)) by 240 and 300 min, respectively. Unstimulated leukocyte oxidative activity, as determined by luminol enhanced chemiluminescence; plasma elastase, a marker of in vivo neutrophil degranulation; and sP-selectin, a marker of endothelial cell activation, did not change throughout. DISCUSSION: Differences in immune cell numbers showed a marked similarity to changes previously reported in response to intense short- and long-duration exercise and were attributed to the physiological responses induced by acute hypoxia that are known to mediate immune cell trafficking. These findings could be relevant to the etiology of conditions where hypoxia and immune cells are implicated.

Observing a fictitious stressful event: haematological changes, including circulating leukocyte activation.

The aim of this study was to assess the effect of watching a psychological stressful event on the activation of leukocytes in healthy human volunteers. Blood samples were obtained from 32 healthy male and female subjects aged between 20 and 26 years before, during and after either watching an 83-minute horror film that none of the subjects had previously seen (The Texas Chainsaw Massacre, 1974) or by sitting quietly in a room (control group). Total differential cell counts, leukocyte activation as measured by the nitroblue tetrazolium (NBT) test, heart rate and blood pressure (BP) measurements were taken at defined time points. There were significant increases in peripheral circulating leukocytes, the number of activated circulating leukocytes, haemoglobin (Hb) concentration and haematocrit (Hct) in response to the stressor. These were accompanied by significant increases in heart rate, systolic and diastolic BP (P<0.05 from baseline). This is the first reported study on the effects of observing a psychologically stressful, albeit fictitious event on circulating leukocyte numbers and the state of leukocyte activation as determined by the nitrotetrazolium test.

Hypoxia-induced chemiluminescence in human leukocytes: the role of Ca2+.

Circumstantial evidence suggests that in vivo hypoxia can activate human leukocytes to release a host of mediators including oxygen free radicals. Endothelial cells have always been presumed to play an important role in hypoxia-induced leukocyte activation. The primary aim of this study was to determine whether acute exposure of whole blood to a hypoxic mixture could stimulate the production of oxygen free radicals in the absence of endothelial cells, and to determine the role of Ca2+ in this process. Freshly drawn whole blood was obtained by venepuncture from 30 healthy volunteers. Exposure of blood to 6% O2 in a purpose built environmental chamber for periods of 8, 10, 15, 30, and 45 min resulted in a significant increase in the production of oxygen free radicals. This response was significantly increased after a 10-min incubation with the Ca2+ ionophore (A23187), supporting the idea that the influx of extracellular Ca2+ is an important step in the process. Incubation of human leukocytes with verapamil (10 min) and subsequent exposure to hypoxia resulted in the significantly reduction in hypoxia-induced production of oxygen free radicals at 15 min and a paradoxical increase at 30- and 45-min exposures. The significance of the results is discussed.