Lesson of the month: novel method to quantify neutrophil uptake in early lung cancer using SPECT-CT.

Neutrophils play an important role in the lung tumour microenvironment. We hypothesised that radiolabelled neutrophils coupled to single-photon emission CT (SPECT) may non-invasively quantify neutrophil uptake in tumours from patients with non-small cell lung cancer. We demonstrated increased uptake of radiolabelled neutrophils from the blood into tumours compared with non-specific uptake using radiolabelled transferrin. Moreover, indium-111-neutrophil activity in the tumour biopsies also correlated with myeloperoxidase (MPO)-positive neutrophils. Our data support the utility of imaging with In-111-labelled neutrophils and SPECT-CT to quantify neutrophil uptake in lung cancer.

Stimulation of the hepatic arterial buffer response using exogenous adenosine: hepatic rest/stress perfusion imaging.

OBJECTIVES: The hepatic arterial buffer response is a mechanism mediated by adenosine whereby hepatic arterial perfusion (HAP) increases when portal flow decreases, and is implicated in liver disease. The first study aim was to measure HAP in patients undergoing myocardial perfusion imaging (MPI), thus developing hepatic arterial rest/stress perfusion imaging (HAPI). The second aim was to compare adenosine-induced changes in splenic perfusion (SP) and HAP with corresponding changes in myocardial blood flow (MBF). METHODS: Patients had MPI with 82Rb PET/CT using adenosine (n = 45) or regadenoson (n = 33) for stressing. SP and HAP were measured using a first-pass technique that gives HAP rather than total hepatic perfusion. Renal perfusion (RP) was also measured. RESULTS: Mean MBF and HAP increased after both adenosine ([stress-rest]/rest 1.1 and 0.8) and regadenoson (1.4 and 0.6), but the respective changes did not correlate. After adenosine, SP (- 0.48) and RP (- 0.26) both decreased. The change in SP correlated positively with the change in MBF (r = 0.36; p = 0.015) but did not correlate with change in HAP. After regadenoson, SP (0.2) and RP (0.2) both increased. The changes in SP correlated with the changes in MBF (r = 0.39; p = 0.025) and HAP (r = 0.39; p = 0.02). Changes in RP correlated with changes in HAP (r = 0.51; p = 0.0008) but not MBF. Resting SP (r = 0.32; p = 0.004), but not resting HAP, correlated with hepatic fat burden. Adenosine-induced change in HAP also correlated with hepatic fat (r = 0.29; p = 0.05). CONCLUSION: HAPI could be a useful new hepatic function test. Neither splenic 'switch-off' nor hepatic arterial 'switch-on' identifies adequacy of stress in MPI. KEY POINTS: • This article describes a new method for assessing arterial perfusion of the liver and its capacity to respond to an infusion of adenosine, a substance that normally 'drives' hepatic arterial flow. • Hepatic arterial flow increased in response to adenosine, sometimes dramatically. Adenosine is already used clinically to stimulate myocardial blood flow in patients with suspected coronary disease, but the increase in flow did not correlate with the corresponding increase in hepatic arterial flow. • Analogous to the use of adenosine in the myocardium, the increase in hepatic arterial flow in response to adenosine has the potential to be a new clinically useful method for the evaluation of hepatic arterial haemodynamics in liver disease.

Use of autologous 99mTechnetium-labelled neutrophils to quantify lung neutrophil clearance in COPD.

RATIONALE: There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung. AIM: To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT). METHODS: 20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with 99mTechnetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis. RESULTS: There was increased accumulation of 99mTc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge. CONCLUSIONS: This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.

Radiolabelled leucocytes in human pulmonary disease.

Introduction: Radionuclides for leucocyte kinetic studies have progressed from non-gamma emitting cell-labelling radionuclides through gamma emitting nuclides that allow imaging of leucocyte kinetics, to the next goal of positron emission tomography (PET). Sources of data: Mostly the authors' own studies, following on from studies of the early pioneers. Areas of controversy: From early imaging studies, it appeared that the majority of the marginated granulocyte pool was located in the lungs. However, later work disputed this by demonstrating the exquisite sensitivity of granulocytes to ex vivo isolation and labelling, and that excessive lung activity is artefactual. Areas of agreement: Following refinement of labelling techniques, it was shown that the majority of marginated granulocytes are located in the spleen and bone marrow. The majority of leucocytes have a pulmonary vascular transit time only a few seconds longer than erythrocytes. The minority showing slow transit, ~5% in healthy persons, is increased in systemic inflammatory disorders that cause neutrophil priming and loss of deformability. Using a range of imaging techniques, including gamma camera imaging, whole-body counting and single photon-emission computerized tomography, labelled granulocytes were subsequently used to image pulmonary trafficking in lobar pneumonia, bronchiectasis, chronic obstructive pulmonary disease and adult respiratory distress syndrome. Growing points: More recently, eosinophils have been separated in pure form using magnetic bead technology for the study of eosinophil trafficking in asthma. Areas timely for developing research: These include advancement of eosinophil imaging, development of monocyte labelling, development of cell labelling with PET tracers and the tracking of lymphocytes.

Glomerular filtration rate: new age- and gender- specific reference ranges and thresholds for living kidney donation.

BACKGROUND: There is a need for a large, contemporary, multi-centre series of measured glomerular filtration rates (mGFR) from healthy individuals to determine age- and gender-specific reference ranges for GFR. We aimed to address this and to use the ranges to provide age- and gender-specific advisory GFR thresholds considered acceptable for living kidney donation. METHODS: Individual-level data including pre-donation mGFR from 2974 prospective living kidney donors from 18 UK renal centres performed between 2003 and 2015 were amalgamated. Age- and gender-specific GFR reference ranges were determined by segmented multiple linear regression and presented as means ± two standard deviations. RESULTS: Males had a higher GFR than females (92.0 vs 88.1 mL/min/1.73m2, P < 0.0001). Mean mGFR was 100 mL/min/1.73m2 until 35 years of age, following which there was a linear decline that was faster in females compared to males (7.7 vs 6.6 mL/min/1.73m2/decade, P = 0.013); 10.5% of individuals aged > 60 years had a GFR < 60 mL/min/1.73m2. The GFR ranges were used along with other published evidence to provide advisory age- and gender-specific GFR thresholds for living kidney donation. CONCLUSIONS: These data suggest that GFR declines after 35 years of age, and the decline is faster in females. A significant proportion of the healthy population over 60 years of age have a GFR < 60 mL/min/1.73m2 which may have implications for the definition of chronic kidney disease. Age and gender differences in normal GFR can be used to determine advisory GFR thresholds for living kidney donation.

Lymphatic drainage efficiency: a new parameter of lymphatic function.

Background Following convection from blood capillaries, plasma proteins are transported to loco-regional lymph nodes in two stages: first, uptake into peripheral lymphatics, and second, transport to nodes. Purpose To introduce a new parameter of lymphatic function that quantifies stage 2 - lymphatic drainage efficiency (LDE). Material and Methods Percentage injected activity (IIQ) in ilio-inguinal nodes 150 min following subcutaneous foot web-space injection of Tc-99 m-nanocolloid was measured in 102 patients undergoing lymphoscintigraphy using a method in which a standard is placed by image guidance over the nodes. Percentage activity leaving the injection depot by 150 min ( k) was measured in 60/102 patients. LDE (%) = 100 × (IIQ/ k). Abnormal lymphoscintigraphy was defined qualitatively as: (i) no activity in ilio-inguinal nodes at 45 min or negligible activity at 150 min (delay); (ii) lymph diversion through skin and/or deep system; and (iii) focal tracer accumulation suggesting cellulitis. Results Scintigraphy was bilaterally normal in 82 limbs, unilaterally normal in 40 limbs and abnormal in 82 limbs. IIQ correlated with k in bilaterally normal (r = 0.86; n = 52), unilaterally normal (r = 0.67; n = 27), and abnormal (r = 0.82; n = 41) limbs. IIQ, k, and LDE were significantly lower in unilaterally normal (9.3 ± 5.4%, 13.8 ± 7.1%, and 65 ± 30%) compared with bilaterally normal limbs (15.4 ± 8.4% [ P > 0.0001], 18.3 ± 8.9% [ P = 0.025], and 84 ± 30% [ P = 0.01]). LDE was lower in limbs displaying skin diversion and/or delay. Conclusion LDE is a new quantitative index that has potential value in clinical research but requires further clinical evaluation. Abnormal quantitative indices indicate that limbs unilaterally normal on lymphoscintigraphy are not functionally normal.

Quantification of tumour (18) F-FDG uptake: Normalise to blood glucose or scale to liver uptake?

PURPOSE: To compare normalisation to blood glucose (BG) with scaling to hepatic uptake for quantification of tumour (18) F-FDG uptake using the brain as a surrogate for tumours. METHODS: Standardised uptake value (SUV) was measured over the liver, cerebellum, basal ganglia, and frontal cortex in 304 patients undergoing (18) F-FDG PET/CT. The relationship between brain FDG clearance and SUV was theoretically defined. RESULTS: Brain SUV decreased exponentially with BG, with similar constants between cerebellum, basal ganglia, and frontal cortex (0.099-0.119 mmol/l(-1)) and similar to values for tumours estimated from the literature. Liver SUV, however, correlated positively with BG. Brain-to-liver SUV ratio therefore showed an inverse correlation with BG, well-fitted with a hyperbolic function (R = 0.83), as theoretically predicted. Brain SUV normalised to BG (nSUV) displayed a nonlinear correlation with BG (R = 0.55); however, as theoretically predicted, brain nSUV/liver SUV showed almost no correlation with BG. Correction of brain SUV using BG raised to an exponential power of 0.099 mmol/l(-1) also eliminated the correlation between brain SUV and BG. CONCLUSION: Brain SUV continues to correlate with BG after normalisation to BG. Likewise, liver SUV is unsuitable as a reference for tumour FDG uptake. Brain SUV divided by liver SUV, however, shows minimal dependence on BG. KEY POINTS: • FDG standard uptake value in tumours helps clinicians assess response to treatment. • SUV is influenced by blood glucose; normalisation to blood glucose is recommended. • An alternative approach is to scale tumour SUV to liver SUV. • The brain used as a tumour surrogate shows that neither approach is valid. • Applying both approaches, however, appropriately corrects for blood glucose.

Pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome.

RATIONALE: Acute respiratory distress syndrome (ARDS) affects over 200000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results mainly from extra-pulmonary organ failure; however the mechanism for this organ crosstalk has not been determined. METHODS: Using autologous radiolabelled neutrophils we investigated the pulmonary transit of primed and unprimed neutrophils in humans. Flow cytometry of whole blood samples was used to assess transpulmonary neutrophil priming gradients in patients with ARDS, sepsis and perioperative controls. MAIN RESULTS: Unprimed neutrophils passed through the lungs with a transit time of 14.2 s, only 2.3 s slower than erythrocytes, and with <5% first-pass retention. Over 97% of neutrophils primed ex vivo with granulocyte macrophage colony-stimulating factor were retained on first pass, with 48% still remaining in the lungs at 40 min. Neutrophils exposed to platelet-activating factor were initially retained but subsequently released such that only 14% remained in the lungs at 40 min. Significant transpulmonary gradients of neutrophil CD62L cell surface expression were observed in ARDS compared with perioperative controls and patients with sepsis. CONCLUSIONS: We demonstrated minimal delay and retention of unprimed neutrophils transiting the healthy human pulmonary vasculature, but marked retention of primed neutrophils; these latter cells then 'deprime' and are re-released into the systemic circulation. Further, we show that this physiological depriming mechanism may fail in patients with ARDS, resulting in increased numbers of primed neutrophils within the systemic circulation. This identifies a potential mechanism for the remote organ damage observed in patients with ARDS.

Use of 111-Indium-labeled autologous eosinophils to establish the in vivo kinetics of human eosinophils in healthy subjects.

Eosinophils are the major cellular effectors of allergic inflammation and represent an important therapeutic target. Although the genesis and activation of eosinophils have been extensively explored, little is known about their intravascular kinetics or physiological fate. This study was designed to determine the intravascular life span of eosinophils, their partitioning between circulating and marginated pools, and sites of disposal in healthy persons. Using autologous, minimally manipulated 111-Indium-labeled leukocytes with blood sampling, we measured the eosinophil intravascular residence time as 25.2 hours (compared with 10.3 hours for neutrophils) and demonstrated a substantial marginated eosinophil pool. γ camera imaging studies using purified eosinophils demonstrated initial retention in the lungs, with early redistribution to the liver and spleen, and evidence of recirculation from a hepatic pool. This work provides the first in vivo measurements of eosinophil kinetics in healthy volunteers and shows that 111-Indium-labeled eosinophils can be used to monitor the fate of eosinophils noninvasively.

Evaluation of non-polynomial equations for one-compartment correction of slope-intercept GFR: theoretical prediction and experimental measurement.

BACKGROUND: Polynomial equations for one-compartment correction of slope-intercept glomerular filtration rate (GFR) will underestimate values at high clearance rates. Non-polynomial correction equations that are independent of patient size and renal function would be advantageous and may have cross-species use. MATERIALS AND METHODS: The study explored the theoretical basis of firstly the Jodal and Brochner-Mortensen one-compartment correction equation, replacing plasma volume with extracellular fluid volume, and secondly an equation described by Peters. One-compartment correction factors (a which is related to plasma volume and v which is related to extracellular fluid volume) which avoided the need for scaling to body size were developed. Both factors were determined from the biexponential clearance curve of the markers iohexol and (51)Cr-EDTA in humans and iohexol in cats and dogs. Relationships between a and v and filtration function and body size were then determined using data from humans, cats and dogs to assess their validity and compare this with theoretical predictions. RESULTS: In all species, v was higher than a, as theoretically predicted. Both were significantly higher in humans than cats and dogs, ruling out cross-species use. Significant relationships were present between v and measures of filtration function in humans, but were weak with respect to a. Neither a nor v showed significant relationships with filtration function in animals or with body size in any species. CONCLUSIONS: a and v (which are factors independent of body size) can be used interchangeably for correcting slope-intercept clearance. However values of both for humans are higher compared to cats and dogs. Therefore a single cross-species factor cannot be used.

Errors in measurement of glomerular filtration rate using the slope-intercept technique and their identification.

BACKGROUND: GFR measured from plasma sampling may be expressed as slope-intercept GFR (SI-GFR) and scaled to body surface area (mGFR/BSA) or as GFR per unit extracellular fluid volume (mGFR/ECV), which is based only on half-time. Measurement errors comprise 3 categories. Pre-injection error arises from error in administered marker and is suspected when mGFR/BSA and mGFR/ECV disagree. Injection errors include 'tissued' injections. Post-injection errors include inaccurate sample timing, inaccurate pipetting, sample haemolysis and sampling through long IV lines through which marker was administered. The aim of the study was to evaluate the impact of errors on mGFR. METHODS: We compared mGFR/BSA with mGFR/ECV in 898 patients undergoing routine investigation. To investigate post-injection error, we took two further patient datasets with r values (correlation coefficient of the 3-sample fit) of 1.0 and introduced errors, in isolation, into each of the 3 recorded sample values, as follows: pipetting (volume) errors of -20%, -10%, -5%, 5%, 10% and 20%, and timing errors of -15 min, -10 min, -5 min, 5 min, 10 min and 15 min. RESULTS: The correlation between mGFR/BSA and mGFR/ECV was close and independent of r. Post-injection error depended on the time of the sample in which it occurred. r correlated poorly with error magnitude for both volume and timing errors. When a 'rogue' sample is suspected its error needed to be substantial for it to be identified by single sample estimates applied to the other samples. CONCLUSION: SI-GFR is resistant to post-injection timing and volume errors but not to pre-injection error.

Noninvasive measurement of tracer extraction efficiency in tissue, illustrated with Tc-99m-MAG3.

OBJECTIVE: The aim of this study is to develop a noninvasive technique for measuring tissue tracer extraction efficiency ( E ) and illustrate it for Tc-99m-mercaptoacetyltriglycine (MAG3) and kidney. METHODS: E was measured in 10 patients with normal MAG3 renography. E is the ratio of tissue clearance-to-blood flow ( Ki/F ). For single-photon tracers, attenuation constants are unknown, so Ki and F cannot be separately measured. However, by deriving attenuation-uncorrected Ki' and F' from the same regions of interests (ROIs), these constants cancel out, giving E . Using a lung ROI for blood activity, F was measured from first-pass and Ki' from Gjedde-Patlak-Rutland (GPR) analysis up to 130 s. Because of interference from right ventricle, a left ventricular ROI (LV) is unsuitable for F' but was used in GPR analysis, making an adjustment for the ratio of respective blood pool signals arising from lung and LV ROIs in early frames (60-90 s). RESULTS: A lung ROI underestimates F' by 4% at normal LV function. Chest wall interstitial activity ( I ), which does not affect F' , amounted to 53 and 30% of the lung and LV signals at 20 min, and 12 and 6% at 130 s, resulting in underestimations of Ki of 4 and 2%, respectively. Ignoring these opposing errors, E based on lung ROI for left and right kidneys was 43.5 (SD 8)% and 47.3 (9)%, and based on LV ROI for GPR analysis was 44.5 (10.9)% and 48.3 (10.6)%. CONCLUSION: E can be measured by combining blood flow from first-pass with clearance from GPR analysis, and has potential value both clinically and in clinical research.

Recycling rate of bile acids in the enterohepatic recirculation as a major determinant of whole body 75SeHCAT retention.

Measurement of the whole body retention of orally administered (75)SeHCAT is used to investigate patients with unexplained diarrhoea. Retention values of <15 % at 7 days post-administration are taken to indicate bile acid malabsorption (BAM). Whilst idiopathic BAM is frequently diagnosed with (75)SeHCAT, functional and morphological studies of the terminal ileum rarely show any abnormality, so the disorder may be more appropriately termed bile acid diarrhoea (BAD). In addition to malabsorption, excess bile acid may reach the colon, where the events leading to diarrhoea take place, as a result firstly of increased bile acid synthesis and secondly of an increased recycling rate of bile acids. Increased recycling has been largely ignored as a cause of BAD, but, as shown in this study, can readily result in excess bile acids reaching the colon even when ileal absorption efficiency is normal (i.e. 95-97 %). There needs to be a re-evaluation of the causes of BAD in patients without a history of previous intestinal resection or evidence of ileal pathology, such as Crohn's disease.

Neutrophil kinetics in health and disease.

Neutrophils play a key role in the elimination of pathogens. They are remarkably short-lived with a circulating half life of 6-8h and hence are produced at a rate of 5x10(10)-10x10(10) cells/day. Tight regulation of these cells is vital because they have significant histotoxic capacity and are widely implicated in tissue injury. This review outlines our current understanding of how neutrophils are released from the bone marrow; in particular, the role of the CXC chemokine receptor 4/stromal-derived factor 1 axis, the relative size and role of the freely circulating and marginated (i.e. slowly transiting) pools within the vascular compartment, and the events that result in the uptake and removal of circulating neutrophils. We also review current understanding of how systemic stress and inflammation affect this finely balanced system.

Higher extracellular fluid volume in women is concealed by scaling to body surface area.

OBJECTIVE: The objective was to assess body surface area (BSA) for scaling extracellular fluid volume (ECV) in comparison with estimated lean body mass (LBM) and total body water (TBW) across a range of body mass indices (BMI). METHODS: This was a multi-centre study from 15 centres that submitted raw data from routine measurement of GFR in potential kidney transplant donors. There were 819 men and 1059 women in total. ECV was calculated from slope-intercept and slope-only measurements of GFR. ECV was scaled using two methods: Firstly, division of ECV by the scaling variable (ratio method), and secondly the regression method of Turner and Reilly. Subjects were placed into five BMI groups: < 20, 20-24.9, 25-29.9, 30-34.9, and 35 + kg/m(2). LBM and TBW were estimated from previously published, gender-specific prediction equations. RESULTS: Ratio and regression scaling gave almost identical results. ECV scaled to BSA by either method was higher in men in all BMI groups but ECV scaled to LBM and TBW was higher in women. There was, however, little difference between men and women in respect to ECV per unit weight in any BMI group, even though women have 10% more adipose tissue. The relations between TBW and BSA and between LBM and BSA, but not between LBM and TBW, were different between men and women. CONCLUSION: Lean tissue in women contains more extracellular water than in men, a difference that is obscured by scaling to BSA. The likely problem with BSA is its insensitivity to body composition.

Quantitative lymphoscintigraphy of the lower limbs for the diagnosis of phlebolymphoedema.

INTRODUCTION: Phlebolymphoedema is caused by the interaction of the venous and lymphatic systems in a state of chronic venous insufficiency in which increased microvascular filtration causes an increased rate of lymph production. Lymphatic drainage rate increases in response, but this is unsustainable and can cause lymphatic failure and oedema. We hypothesise that in phlebolymphoedema we could measure unusually high lymphatic drainage while the lymph system is still fully functional. METHOD: Patients referred for lymphoscintigraphic investigation of swollen legs between April 2021 and December 2022 were reviewed. Quantitative lymphoscintigraphy was performed following the technique of Keramida et al . (2017) and ilio-inguinal nodal uptake (IIQ%) was calculated. The presence of scintigraphic features of increased lymph production was noted for each limb. RESULTS: A total of 39 patients were reviewed (78 limbs, 29F, 10M). Seven limbs were identified with supranormal lymphatic function (IIQ > 30%) plus three borderline. Of these 10 limbs, all had at least two scintigraphic features of increased lymph production. CONCLUSION: Quantitative lymphoscintigraphy, although developed for diagnosing abnormally low lymphatic function, may also have utility at the upper end of the spectrum for identifying chronic venous insufficiency. An IIQ% upper normal limit of 30% could be used to diagnose venous insufficiency as the cause for limb swelling. This is of note for patients of large body habitus in whom venous ultrasound is difficult.

Acute lung injury results from failure of neutrophil de-priming: a new hypothesis.

Neutrophils are the most abundant circulating white cell in humans and play a crucial role in the innate immune response. Accumulation and activation of neutrophils, together with delayed clearance, have been shown to be a key event in the pathogenesis of acute lung injury. Previously, it has been proposed that there is substantial pooling of neutrophils within the pulmonary vasculature, even under physiological conditions, making the lung especially vulnerable to neutrophil-mediated tissue injury. However, more recent evidence suggests that only primed neutrophils accumulate in the pulmonary vasculature. This article examines the evidence for these two opposing views and proposes a new two-step model for the recruitment of neutrophils into the lung. Firstly, neutrophils that become primed, by exposure to a range of inflammatory mediators or physicochemical perturbations, become shape changed and stiff because of alterations in their cytoskeleton, and as a result, accumulate within the pulmonary circulation. In the absence of further stimuli, the healthy pulmonary vasculature is able to selectively retained these primed cells, allow them to 'de-prime' and be released back into the circulation in a quiescent, state. If this pulmonary 'de-priming' mechanism fails, or a second insult occurs, such as ventilator-associated barotrauma, which causes loss of alveolar integrity, primed neutrophils migrate from the pulmonary vasculature into the interstitial space with resultant lung injury. This canonical 'two step' model highlights the importance of neutrophil priming in the genesis of lung injury and the importance of adopting strategies to minimise alveolar injury.