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Serum neutrophil gelatinase–associated lipocalin in ballistic injuries: A comparison between blast injuries and gunshot wounds
Journal of Critical Care (2012) 27, 419.e1–419.e5
Trauma
Serum neutrophil gelatinase–associated lipocalin in ballistic injuries: A comparison between blast injuries and gunshot wounds☆ Adrian J. Mellor FRCA a, b,⁎, David Woods MRCP, MD c, d, e, f a
Surg Cdr RN, Defence Medical Services James Cook University Hospital, Middlesbrough, TS4 3 BW c Lt Col, RAMC, Defence Medical Services d Newcastle and Northumbria NHS Trusts e Wansbeck General and Royal Victoria Infirmary f University of Newcastle b
Keywords: NGAL; Outcome; Blast injury
Abstract Neutrophil gelatinase–associated lipocalin (NGAL) is part of a functionally diverse family of proteins that generally bind small, hydrophobic ligands. Neutrophil gelatinase–associated lipocalin is expressed in a number of human tissues including gastrointestinal, respiratory, and urinary tracts and tends to rise in response to inflammation. For this reason, we hypothesized that levels of NGAL might be expressed at higher levels after blast injury compared with other ballistic injury. Purpose: The purpose of this study is to test the hypothesis that NGAL may be a marker of injury severity in blast injury. Materials: Twenty-three combat casualties (13 blast, 10 gunshot wounds) admitted to the multinational role 3 facility in Helmand province were studied. Serum NGAL was measured using a Biosite Triage point-of-care monitor at 5 time points after injury. Results: Neutrophil gelatinase–associated lipocalin rose in both groups of casualties and was significantly predictive of death or renal failure at intensive care unit admission, 12 and 24 hours after injury. Conclusions: Neutrophil gelatinase–associated lipocalin is not a specific marker of blast injury but is predictive of both renal failure and poor outcome. © 2012 Elsevier Inc. All rights reserved.
1. Introduction ☆
Neither author has any conflict of interest to declare. Alere Ltd unconditionally provided NGAL test cartridges at a reduced cost. The authors would like to acknowledge the assistance of Dr Simon Turner in preparing the manuscript for publication. ⁎ Corresponding author. E-mail address: [email protected] (A.J. Mellor). 0883-9441/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jcrc.2011.08.019
Neutrophil gelatinase–associated lipocalin (NGAL) is part of a functionally diverse family of proteins that generally bind small, hydrophobic ligands. Neutrophil gelatinase–associated lipocalin is expressed in a number
419.e2 of human tissues including gastrointestinal, respiratory, and urinary tracts and tends to rise in response to inflammation [1]. One interesting property of the protein is its siderophore-chelating property, and it acts as a component of immunity to exogenous bacterial and fungal infections, depleting the intracellular iron stores of the microorganisms. Iron is an essential nutrient for bacterial and fungal organisms, and this ability to compete for iron stores by scavenging microbial siderophores (iron chelators) is a useful defense against microbial infection [2,3]. Increases in systemic and tissue NGAL expression have been well documented in many conditions characterized by infection or inflammation, including diverticulitis, appendicitis, inflammatory bowel disease, or urinary tract infection, but clinical interest has mainly focused on use in monitoring both acute and chronic renal failure [4]. In particular, it may be of use in diagnosing acute kidney injury (AKI) in the setting of cardiac surgery and contrast-related nephropathy [5-7]. The upper limit of normal for serum NGAL is 149 ng/mL with values greater than 204 ng/mL associated with AKI in nonseptic patients (values N293 ng/mL in septic patients) [8]. Data from the use of NGAL in the intensive care setting suggest that NGAL is a sensitive (but not specific) indicator of AKI, depending upon the nature of the critical illness [8,9]. Part of the problem with the marker in this patient group is the fact that many patients may have preexisting renal impairment and also that NGAL may merely be a marker of acute inflammation [10]. Blast injury is caused when shock waves and blast winds, created by an explosion, meet the human body. Primary blast injury is due to the sudden increase in air pressures and damages gas containing spaces. The damage created depends on the pressure changes and, as a consequence, is greater closer to the explosion. Trauma to the eardrum occurs at low pressures (about 2 psi), whereas blast lung requires much greater pressures (70 psi), pressures greater than 80 psi carry a greater than 50% mortality [11]. Primary blast injury occurs within the brain, lung, and gastrointestinal tract as a result of shear forces imparted by the shock wave at a tissue/gas interface. As a result, it could be speculated that the multisystem insult (and specifically the lung and gastrointestinal damage) represented by blast injury would cause a proportionally greater rise in NGAL when compared with gunshot wounds (GSW). In this way, greater elevations in NGAL may reflect greater tissue damage and potentially relate to mortality from otherwise invisible primary blast damage. We hypothesized that serum NGAL may be a practical and effective marker of outcome rising with increasing severity of blast injury. The care of injuries from blast often takes place in relatively austere conditions and with limited capability for treatment of casualties with organ system failures. The utility of a measure of severity of injury would be to triage patients and/or expedite retrieval to areas where a higher level of care, such as extracorporeal membrane oxygenation, is available.
A.J. Mellor, D. Woods
2. Method Neutrophil gelatinase–associated lipocalin assays are routinely used in some centers as an early warning of AKI, and as the assay was to be performed on blood taken routinely in the clinical care of patients in the hospital, it was deemed by the Ministry of Defence Research Ethics Committee (MODREC, UK) that this work could be carried out as part of clinical audit and did not, therefore, require formal ethical approval. Consecutive trauma patients were studied at the UK Role 3 Hospital Facility, Camp Bastion, Helmand Province, Afghanistan. Adult patients were included if they met the following criteria: 1. Trauma team activation 2. Resuscitation with blood products in the emergency department 3. Requirement for treatment post operatively on the intensive care unit (ICU) Blood was collected in an EDTA blood collection tube at 5 time points: 1. 2. 3. 4. 5.
On admission (NGAL1) After 60 minutes of resuscitation (NGAL2) On admission to the ICU (NGAL3) Twelve hours after admission (NGAL4) Twenty-four hours after admission (NGAL5)
Blood was then analyzed on a Biosite Triage point of care monitor (Alere Medical, Stockport, UK) using a Triage NGAL test kit, a point-of-care, fluorescence-based immunoassay. This gives a rapid quantitative measurement of NGAL concentration in EDTA anticoagulated whole blood or plasma specimens. The assay device is a singleuse plastic cartridge that contains an NGAL-specific monoclonal antibody conjugated to a fluorescent NGAL antigen immobilized on a solid phase. The test cartridge includes positive and negative control immunoassays; these are performed for each test and reported as an error if abnormal. The test is performed by pipetting several drops of whole blood or plasma onto a port in the cassette where a filter separates cells from plasma. The plasma then reconstitutes the fluorescent antibody and flows down the diagnostic lane via capillary action. Neutrophil gelatinase– associated lipocalin present in the specimen prevents binding of the fluorescent detection particles to the solid phase immobilized in the detection zone, such that the NGAL concentration is inversely proportional to the fluorescence detected. Separate solid phase zones are located along the same diagnostic lane for the control assay systems. The cassette is then inserted into the Triage Meter, a portable fluorescence spectrometer, and quantitative measurements of NGAL concentration in the range from 60 to 1300 ng/mL are displayed on the meter screen.
Serum NGAL in ballistic injuries
419.e3 NGAL at each time point studied 800 700 600 NGAL (ng/ml)
Clinical data including patient demographics, mechanism of injury, arterial blood gas measurements, serum creatinine, blood products transfused, ICU length of stay, diagnosis of acute renal failure (defined as urine output of b0.3 mL/kg for 24 hours, a tripling in serum creatinine or a requirement for renal replacement therapy) [12] were recorded.
500 400 300 200
3. Statistical analysis
100
For statistical calculations, the software package SPSS 14.0 was used. Not all subjects had complete sets of data for all 5 time points, but all subjects were included in the analysis. Reasons for incomplete data sets were death before the fifth sampling time, transfer out to another facility or where routine bloods were not taken (in accordance with ethical committee permissions). All data were tested for Gaussian distribution using the Kolmogorov-Smirnov test and Shapiro Wilks statistic. If distribution was not Gaussian (eg, NGAL), the nonparametric Mann Whitney U test was used when independent groups were concerned. For paired, dependent samples (eg, NGAL on admission vs 12 hours later), the nonparametric Wilcoxon signed rank test was used. A Friedman test was used for the repeated measures of NGAL over all time points. Correlation analyses for nonparametric data were done using Spearman correlation coefficient. For the analysis of dependent variables that were normally distributed (eg, base excess), changes were tested by the paired Student t test. For independent variables that were normally distributed an independent-samples t test was used. Correlation analysis for normally distributed data was performed by calculating the Pearson coefficient of correlation. P b .05 (2 sided) was considered significant.
4. Results Twenty-three male patients were studied, and the mean age was 29.8 years (range, 18-60 years); mean time from injury to arrival in resuscitation area of field hospital was 60.6 minutes (range, 20-170 minutes). Of these, 10 had been injured by GSW, and 13, by blast injuries. Table 1 shows the presenting blood pressure, oxygen saturation and heart rate, and the number of units of red blood cells transfused during resuscitation and initial surgical procedure. The initial base excess was −10.6 ± 7.8 (mean ± Table 1
0 NGAL1
NGAL2
NGAL3
NGAL4
NGAL5
Fig. 1 Neutrophil gelatinase–associated lipocalin at each time point. Neutrophil gelatinase–associated lipocalin values were significantly different between time points 1 and 2 (P = .021), where a significant fall occurred. Between time points 2 and 3 (P = .006), time points 2 and 5 (P = .037), and time points 4 and 5 (P = .028), significant rises in NGAL occurred. Data are presented as mean ± SD.
SD) in blast injuries vs −5.6 ± 5.7 (mean ± SD) in GSW; this was not statistically significant. Patients with blast injury required significantly more blood during resuscitation and initial surgical procedure than those with GSW (10.2 ± 3.1 vs 6.9 ± 1.8, mean ± SD; P = .006). There were no other significant differences in the presenting parameters by injury. Repeated measures analysis demonstrated a significant change in NGAL over time (P = .04). Neutrophil gelatinase– associated lipocalin values were significantly different between time points 1 and 2 (P = .021) where a significant fall occurred. Between time points 2 and 3 (P = .006), time points 2 and 5 (P = .037), and time points 4 and 5 (P = .028), significant rises in NGAL occurred (Fig. 1). There was no difference in NGAL depending on mechanism of injury (Fig. 2). There was no difference in NGAL at time points 1 and 2 in the 3 subjects that subsequently developed renal failure, but NGAL was significantly higher at time points 3, 4, and 5 in those that developed renal failure vs those that did not: 352 (244-729) vs 151 (90-501); 553 (512-594) vs 177 (60-422); 896 (860-931) vs 279 (60-643) (median [range] P = .037; 0.031 and 0.037 for difference in NGAL in those with subsequent renal failure vs those without at time points 3, 4, and 5, respectively) (Fig. 3). Neutrophil gelatinase–associated lipocalin (nanograms per millimeter) between the survivors and those that died (n = 2) was no different at time points 1 and 2 but was
Presenting physiologic data and transfusion volumes
Mechanism
Systolic BP mm Hg (mean ± SD)
SpO2 % (mean ± SD)
HR (mean ± SD)
BE mmol/L (mean ± SD)
Units of packed RBC transfused (mean ± SD)
GSW Blast
104 ± 28.7 106.9 ± 26.3
97.4 ± 4.8 98.8 ± 2.3
113.8 ± 22.6 122 ± 23.1
-5.6 ± 5.7 -10.6 ± 7.8
6.9 ± 1.8 10.2 ± 3.1
Patients with blast injury required significantly more blood during resuscitation and initial surgical procedure than those with GSW (10.2 ± 3.1 vs 6.9 ± 1.8, mean ± SD; P = .006). There were no other significant differences in the presenting parameters by injury.
419.e4
A.J. Mellor, D. Woods NGAL and mortality 1000 900
900 800 700 600 500 400 300 200 100 0
800
GSW IED
NGAL (ng/ml)
NGAL (ng/ml)
NGAL at each time-point according to mechanism of injury
700 600
Died Survived
500 400 300 200
NGAL 1
NGAL 2
NGAL 3
NGAL 4
100
NGAL 5
0
Fig. 2 Neutrophil gelatinase–associated lipocalin by mechanism of injury. There was no difference in NGAL depending on mechanism of injury (all data, mean ± SD).
significantly different at time points 3, 4, and 5: 194 (60501) vs 541 (352-729); 167 (60-422) vs 553 (512-594); 279 (60-643) vs 896 (860-931) at time points 3, 4, and 5, respectively (median [range] P = .035, P = .028, and P = .037, respectively) (Fig. 4). There were no significant correlations between NGAL and for time from wounding to arrival at hospital, white cell count or SpO2. There were several correlations between, for example, NGAL at time point 4 and pH at time point 3 (ρ −0.845, P ≤ .001) or NGAL at time point 5 and pH3 (ρ −0.807, P = .003), suggesting that the rise in NGAL was reflecting the acid base status on arrival at the ICU. Our data do not allow us to fully explore this possibility.
5. Discussion All the patients had hypovolemic shock on arrival represent a unique series. Prehospital interventions performed during transfer to the hospital in Camp Bastion include rapid sequence induction of anesthesia, endotracheal intubation, and blood transfusion. This explains why only 1 patient (who was rapidly transferred by a nonphysician led prehospital team) had low oxygen saturation on arrival. The patients were all fighting age
NGAL 3
NGAL 4
NGAL 5
Fig. 4 Neutrophil gelatinase–associated lipocalin and mortality (n = 21 survived, 2 died). Neutrophil gelatinase–associated lipocalin by mortality. All data are expressed as mean ± SD.
males and, as such, had no preexisting pathology likely to lead to confounding rises in NGAL. Unfortunately, much of the data collected was incomplete at least in terms of the number of samples collected. This is due to the nature of the medical care in Helmand province, UK, and US casualties are rapidly repatriated for ongoing care, and Afghan casualties are transferred into their own health care system as soon as possible (this is in practice once there is no ongoing requirement for critical care). Although the patterns of injury were all due to ballistic trauma, the individual organs or body regions injured differed from patient to patient. Numbers were too small to allow subgroup analysis of casualties (eg, those with chest, gut, or peripheral injuries), but there was no gross difference in the data. It is interesting to note the initial fall in NGAL between arrival in the hospital and after 60 minutes of resuscitation; we suspect this may be secondary to a dilution effect from transfusion. Despite the limitations of this study, several interesting findings are worthy of further investigation.
5.1. NGAL did not rise in blast to any greater extent than in GSWs
NGAL and Renal Failure 1000 900
NGAL (ng/ml)
800 700 600 500
RF No RF
400 300 200 100 0 NGAL 3
NGAL 4
NGAL 5
Fig. 3 Changes in NGAL with respect to development of renal failure (n = 20, no renal failure, 3 renal failure). All data are expressed as mean ± SD. RF indicates renal failure.
This is an important finding suggesting that mechanism of ballistic injury does not affect NGAL and, therefore, NGAL may have utility as an independent marker of AKI in the military intensive care setting. For reasons outlined above, it could be hypothesized that NGAL would rise with primary blast injury as the protein is expressed by both the gut and the lungs. This does not appear to be the case.
5.2. NGAL levels on admission to ITU and 12 and 24 hours later were significantly greater in those that subsequently died Neutrophil gelatinase–associated lipocalin levels rose to those associated with the development of renal failure [8],
Serum NGAL in ballistic injuries but in our series, renal failure was uncommon. Our findings suggest that in this group of fit, young patients, NGAL rise is related not just to the degree of renal insult but also perhaps the overall systemic inflammatory response.
5.3. NGAL did not rise immediately after injury In fact, it dropped significantly, possibly due to dilution, and only started to rise after arrival on the ITU. This finding is significant and conflicts with other investigators who have suggested NGAL rises within minutes [13] and may be useful as a triage tool in the emergency department [14]. The casualties in this series were all young and previously in very good health, and it may well be that there is a difference between the rapid renal insult of hypovolemia in trauma and the more insidious onset with sepsis or many of the disease states presenting to the emergency department. Our findings, with a rise in NGAL occurring after ICU admission, may reflect the release of NGAL as part of ischemia/reperfusion injury to the nephron or may simply represent the dilution of NGAL as shed blood is replaced with allogenic blood transfusion. In conclusion, although we consider our findings preliminary, an NGAL measurement in trauma cases on arrival at the ICU may prove to be a useful indicator not just of subsequent renal dysfunction but also of mortality. This possibility requires further investigation.
References [1] Kjeldsen L, Johnsen AH, Sengelov H, et al. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem 1993;268:10425-32.
419.e5 [2] Goetz DH, Holmes MA, Borregaard N, et al. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophoremediated iron acquisition. Mol Cell 2002;10:1033-43. [3] Borregaard N, Cowland JB. Neutrophil gelatinase associated lipocalin, a siderophore-binding eukaryotic protein. Biometals 2006;19:211-5. [4] Bolignano D, Donato V, Coppolino G, et al. Neutrophil gelatinase– associated lipocalin (NGAL) as a marker of kidney damage. Am J Kidney Dis 2008;52:595-605. [5] Hirsch R, Dent C, Pfriem H, et al. NGAL is an early predictive biomarker of contrast-induced nephropathy in children. Pediatr Nephrol 2007;22:2089-95. [6] Ling W, Zhaohui N, Ben H, et al. Urinary IL-18 and NGAL as early predictive biomarkers in contrast-induced nephropathy after coronary angiography. Nephron Clin Pract 2008;108:176-81. [7] Mishra J, Dent C, Tarabishi R, et al. Neutrophil gelatinase–associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005;365:1231-8. [8] Bagshaw SM, Bennett M, Haase M, et al. Plasma and urine neutrophil gelatinase–associated lipocalin in septic versus non-septic acute kidney injury in critical illness. Intensive Care Med 2010;36:452-61. [9] Wheeler DS, Devarajan P, Ma Q, et al. Serum neutrophil gelatinase– associated lipocalin (NGAL) as a marker of acute kidney injury in critically ill children with septic shock. Crit Care Med 2008;36: 1297-303. [10] Xu S, Venge P. Lipocalins as biochemical markers of disease. Biochim Biophys Acta 2000;1482:298-307. [11] Mellor SG, Cooper GJ. Analysis of 828 servicemen killed or injured by explosion in Northern Ireland 1970-84: the Hostile Action Casualty System. Br J Surg 1989;76:1006-10. [12] Bellomo R, Ronco C, Kellum JA, et al. Acute renal failure— definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Crit Care 2004;8:R204-12. [13] Haase M, Bellomo R, Haase-Fielitz A. Neutrophil gelatinase– associated lipocalin. Curr Opin Crit Care 2010. [14] Di Grande A, Giuffrida C, Carpinteri G, Narbone G, et al. Neutrophil gelatinase–associated lipocalin: a novel biomarker for the early diagnosis of acute kidney injury in the emergency department. Eur Rev Med Pharmacol Sci 2009;13:197-200.
Trauma
Serum neutrophil gelatinase–associated lipocalin in ballistic injuries: A comparison between blast injuries and gunshot wounds☆ Adrian J. Mellor FRCA a, b,⁎, David Woods MRCP, MD c, d, e, f a
Surg Cdr RN, Defence Medical Services James Cook University Hospital, Middlesbrough, TS4 3 BW c Lt Col, RAMC, Defence Medical Services d Newcastle and Northumbria NHS Trusts e Wansbeck General and Royal Victoria Infirmary f University of Newcastle b
Keywords: NGAL; Outcome; Blast injury
Abstract Neutrophil gelatinase–associated lipocalin (NGAL) is part of a functionally diverse family of proteins that generally bind small, hydrophobic ligands. Neutrophil gelatinase–associated lipocalin is expressed in a number of human tissues including gastrointestinal, respiratory, and urinary tracts and tends to rise in response to inflammation. For this reason, we hypothesized that levels of NGAL might be expressed at higher levels after blast injury compared with other ballistic injury. Purpose: The purpose of this study is to test the hypothesis that NGAL may be a marker of injury severity in blast injury. Materials: Twenty-three combat casualties (13 blast, 10 gunshot wounds) admitted to the multinational role 3 facility in Helmand province were studied. Serum NGAL was measured using a Biosite Triage point-of-care monitor at 5 time points after injury. Results: Neutrophil gelatinase–associated lipocalin rose in both groups of casualties and was significantly predictive of death or renal failure at intensive care unit admission, 12 and 24 hours after injury. Conclusions: Neutrophil gelatinase–associated lipocalin is not a specific marker of blast injury but is predictive of both renal failure and poor outcome. © 2012 Elsevier Inc. All rights reserved.
1. Introduction ☆
Neither author has any conflict of interest to declare. Alere Ltd unconditionally provided NGAL test cartridges at a reduced cost. The authors would like to acknowledge the assistance of Dr Simon Turner in preparing the manuscript for publication. ⁎ Corresponding author. E-mail address: [email protected] (A.J. Mellor). 0883-9441/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jcrc.2011.08.019
Neutrophil gelatinase–associated lipocalin (NGAL) is part of a functionally diverse family of proteins that generally bind small, hydrophobic ligands. Neutrophil gelatinase–associated lipocalin is expressed in a number
419.e2 of human tissues including gastrointestinal, respiratory, and urinary tracts and tends to rise in response to inflammation [1]. One interesting property of the protein is its siderophore-chelating property, and it acts as a component of immunity to exogenous bacterial and fungal infections, depleting the intracellular iron stores of the microorganisms. Iron is an essential nutrient for bacterial and fungal organisms, and this ability to compete for iron stores by scavenging microbial siderophores (iron chelators) is a useful defense against microbial infection [2,3]. Increases in systemic and tissue NGAL expression have been well documented in many conditions characterized by infection or inflammation, including diverticulitis, appendicitis, inflammatory bowel disease, or urinary tract infection, but clinical interest has mainly focused on use in monitoring both acute and chronic renal failure [4]. In particular, it may be of use in diagnosing acute kidney injury (AKI) in the setting of cardiac surgery and contrast-related nephropathy [5-7]. The upper limit of normal for serum NGAL is 149 ng/mL with values greater than 204 ng/mL associated with AKI in nonseptic patients (values N293 ng/mL in septic patients) [8]. Data from the use of NGAL in the intensive care setting suggest that NGAL is a sensitive (but not specific) indicator of AKI, depending upon the nature of the critical illness [8,9]. Part of the problem with the marker in this patient group is the fact that many patients may have preexisting renal impairment and also that NGAL may merely be a marker of acute inflammation [10]. Blast injury is caused when shock waves and blast winds, created by an explosion, meet the human body. Primary blast injury is due to the sudden increase in air pressures and damages gas containing spaces. The damage created depends on the pressure changes and, as a consequence, is greater closer to the explosion. Trauma to the eardrum occurs at low pressures (about 2 psi), whereas blast lung requires much greater pressures (70 psi), pressures greater than 80 psi carry a greater than 50% mortality [11]. Primary blast injury occurs within the brain, lung, and gastrointestinal tract as a result of shear forces imparted by the shock wave at a tissue/gas interface. As a result, it could be speculated that the multisystem insult (and specifically the lung and gastrointestinal damage) represented by blast injury would cause a proportionally greater rise in NGAL when compared with gunshot wounds (GSW). In this way, greater elevations in NGAL may reflect greater tissue damage and potentially relate to mortality from otherwise invisible primary blast damage. We hypothesized that serum NGAL may be a practical and effective marker of outcome rising with increasing severity of blast injury. The care of injuries from blast often takes place in relatively austere conditions and with limited capability for treatment of casualties with organ system failures. The utility of a measure of severity of injury would be to triage patients and/or expedite retrieval to areas where a higher level of care, such as extracorporeal membrane oxygenation, is available.
A.J. Mellor, D. Woods
2. Method Neutrophil gelatinase–associated lipocalin assays are routinely used in some centers as an early warning of AKI, and as the assay was to be performed on blood taken routinely in the clinical care of patients in the hospital, it was deemed by the Ministry of Defence Research Ethics Committee (MODREC, UK) that this work could be carried out as part of clinical audit and did not, therefore, require formal ethical approval. Consecutive trauma patients were studied at the UK Role 3 Hospital Facility, Camp Bastion, Helmand Province, Afghanistan. Adult patients were included if they met the following criteria: 1. Trauma team activation 2. Resuscitation with blood products in the emergency department 3. Requirement for treatment post operatively on the intensive care unit (ICU) Blood was collected in an EDTA blood collection tube at 5 time points: 1. 2. 3. 4. 5.
On admission (NGAL1) After 60 minutes of resuscitation (NGAL2) On admission to the ICU (NGAL3) Twelve hours after admission (NGAL4) Twenty-four hours after admission (NGAL5)
Blood was then analyzed on a Biosite Triage point of care monitor (Alere Medical, Stockport, UK) using a Triage NGAL test kit, a point-of-care, fluorescence-based immunoassay. This gives a rapid quantitative measurement of NGAL concentration in EDTA anticoagulated whole blood or plasma specimens. The assay device is a singleuse plastic cartridge that contains an NGAL-specific monoclonal antibody conjugated to a fluorescent NGAL antigen immobilized on a solid phase. The test cartridge includes positive and negative control immunoassays; these are performed for each test and reported as an error if abnormal. The test is performed by pipetting several drops of whole blood or plasma onto a port in the cassette where a filter separates cells from plasma. The plasma then reconstitutes the fluorescent antibody and flows down the diagnostic lane via capillary action. Neutrophil gelatinase– associated lipocalin present in the specimen prevents binding of the fluorescent detection particles to the solid phase immobilized in the detection zone, such that the NGAL concentration is inversely proportional to the fluorescence detected. Separate solid phase zones are located along the same diagnostic lane for the control assay systems. The cassette is then inserted into the Triage Meter, a portable fluorescence spectrometer, and quantitative measurements of NGAL concentration in the range from 60 to 1300 ng/mL are displayed on the meter screen.
Serum NGAL in ballistic injuries
419.e3 NGAL at each time point studied 800 700 600 NGAL (ng/ml)
Clinical data including patient demographics, mechanism of injury, arterial blood gas measurements, serum creatinine, blood products transfused, ICU length of stay, diagnosis of acute renal failure (defined as urine output of b0.3 mL/kg for 24 hours, a tripling in serum creatinine or a requirement for renal replacement therapy) [12] were recorded.
500 400 300 200
3. Statistical analysis
100
For statistical calculations, the software package SPSS 14.0 was used. Not all subjects had complete sets of data for all 5 time points, but all subjects were included in the analysis. Reasons for incomplete data sets were death before the fifth sampling time, transfer out to another facility or where routine bloods were not taken (in accordance with ethical committee permissions). All data were tested for Gaussian distribution using the Kolmogorov-Smirnov test and Shapiro Wilks statistic. If distribution was not Gaussian (eg, NGAL), the nonparametric Mann Whitney U test was used when independent groups were concerned. For paired, dependent samples (eg, NGAL on admission vs 12 hours later), the nonparametric Wilcoxon signed rank test was used. A Friedman test was used for the repeated measures of NGAL over all time points. Correlation analyses for nonparametric data were done using Spearman correlation coefficient. For the analysis of dependent variables that were normally distributed (eg, base excess), changes were tested by the paired Student t test. For independent variables that were normally distributed an independent-samples t test was used. Correlation analysis for normally distributed data was performed by calculating the Pearson coefficient of correlation. P b .05 (2 sided) was considered significant.
4. Results Twenty-three male patients were studied, and the mean age was 29.8 years (range, 18-60 years); mean time from injury to arrival in resuscitation area of field hospital was 60.6 minutes (range, 20-170 minutes). Of these, 10 had been injured by GSW, and 13, by blast injuries. Table 1 shows the presenting blood pressure, oxygen saturation and heart rate, and the number of units of red blood cells transfused during resuscitation and initial surgical procedure. The initial base excess was −10.6 ± 7.8 (mean ± Table 1
0 NGAL1
NGAL2
NGAL3
NGAL4
NGAL5
Fig. 1 Neutrophil gelatinase–associated lipocalin at each time point. Neutrophil gelatinase–associated lipocalin values were significantly different between time points 1 and 2 (P = .021), where a significant fall occurred. Between time points 2 and 3 (P = .006), time points 2 and 5 (P = .037), and time points 4 and 5 (P = .028), significant rises in NGAL occurred. Data are presented as mean ± SD.
SD) in blast injuries vs −5.6 ± 5.7 (mean ± SD) in GSW; this was not statistically significant. Patients with blast injury required significantly more blood during resuscitation and initial surgical procedure than those with GSW (10.2 ± 3.1 vs 6.9 ± 1.8, mean ± SD; P = .006). There were no other significant differences in the presenting parameters by injury. Repeated measures analysis demonstrated a significant change in NGAL over time (P = .04). Neutrophil gelatinase– associated lipocalin values were significantly different between time points 1 and 2 (P = .021) where a significant fall occurred. Between time points 2 and 3 (P = .006), time points 2 and 5 (P = .037), and time points 4 and 5 (P = .028), significant rises in NGAL occurred (Fig. 1). There was no difference in NGAL depending on mechanism of injury (Fig. 2). There was no difference in NGAL at time points 1 and 2 in the 3 subjects that subsequently developed renal failure, but NGAL was significantly higher at time points 3, 4, and 5 in those that developed renal failure vs those that did not: 352 (244-729) vs 151 (90-501); 553 (512-594) vs 177 (60-422); 896 (860-931) vs 279 (60-643) (median [range] P = .037; 0.031 and 0.037 for difference in NGAL in those with subsequent renal failure vs those without at time points 3, 4, and 5, respectively) (Fig. 3). Neutrophil gelatinase–associated lipocalin (nanograms per millimeter) between the survivors and those that died (n = 2) was no different at time points 1 and 2 but was
Presenting physiologic data and transfusion volumes
Mechanism
Systolic BP mm Hg (mean ± SD)
SpO2 % (mean ± SD)
HR (mean ± SD)
BE mmol/L (mean ± SD)
Units of packed RBC transfused (mean ± SD)
GSW Blast
104 ± 28.7 106.9 ± 26.3
97.4 ± 4.8 98.8 ± 2.3
113.8 ± 22.6 122 ± 23.1
-5.6 ± 5.7 -10.6 ± 7.8
6.9 ± 1.8 10.2 ± 3.1
Patients with blast injury required significantly more blood during resuscitation and initial surgical procedure than those with GSW (10.2 ± 3.1 vs 6.9 ± 1.8, mean ± SD; P = .006). There were no other significant differences in the presenting parameters by injury.
419.e4
A.J. Mellor, D. Woods NGAL and mortality 1000 900
900 800 700 600 500 400 300 200 100 0
800
GSW IED
NGAL (ng/ml)
NGAL (ng/ml)
NGAL at each time-point according to mechanism of injury
700 600
Died Survived
500 400 300 200
NGAL 1
NGAL 2
NGAL 3
NGAL 4
100
NGAL 5
0
Fig. 2 Neutrophil gelatinase–associated lipocalin by mechanism of injury. There was no difference in NGAL depending on mechanism of injury (all data, mean ± SD).
significantly different at time points 3, 4, and 5: 194 (60501) vs 541 (352-729); 167 (60-422) vs 553 (512-594); 279 (60-643) vs 896 (860-931) at time points 3, 4, and 5, respectively (median [range] P = .035, P = .028, and P = .037, respectively) (Fig. 4). There were no significant correlations between NGAL and for time from wounding to arrival at hospital, white cell count or SpO2. There were several correlations between, for example, NGAL at time point 4 and pH at time point 3 (ρ −0.845, P ≤ .001) or NGAL at time point 5 and pH3 (ρ −0.807, P = .003), suggesting that the rise in NGAL was reflecting the acid base status on arrival at the ICU. Our data do not allow us to fully explore this possibility.
5. Discussion All the patients had hypovolemic shock on arrival represent a unique series. Prehospital interventions performed during transfer to the hospital in Camp Bastion include rapid sequence induction of anesthesia, endotracheal intubation, and blood transfusion. This explains why only 1 patient (who was rapidly transferred by a nonphysician led prehospital team) had low oxygen saturation on arrival. The patients were all fighting age
NGAL 3
NGAL 4
NGAL 5
Fig. 4 Neutrophil gelatinase–associated lipocalin and mortality (n = 21 survived, 2 died). Neutrophil gelatinase–associated lipocalin by mortality. All data are expressed as mean ± SD.
males and, as such, had no preexisting pathology likely to lead to confounding rises in NGAL. Unfortunately, much of the data collected was incomplete at least in terms of the number of samples collected. This is due to the nature of the medical care in Helmand province, UK, and US casualties are rapidly repatriated for ongoing care, and Afghan casualties are transferred into their own health care system as soon as possible (this is in practice once there is no ongoing requirement for critical care). Although the patterns of injury were all due to ballistic trauma, the individual organs or body regions injured differed from patient to patient. Numbers were too small to allow subgroup analysis of casualties (eg, those with chest, gut, or peripheral injuries), but there was no gross difference in the data. It is interesting to note the initial fall in NGAL between arrival in the hospital and after 60 minutes of resuscitation; we suspect this may be secondary to a dilution effect from transfusion. Despite the limitations of this study, several interesting findings are worthy of further investigation.
5.1. NGAL did not rise in blast to any greater extent than in GSWs
NGAL and Renal Failure 1000 900
NGAL (ng/ml)
800 700 600 500
RF No RF
400 300 200 100 0 NGAL 3
NGAL 4
NGAL 5
Fig. 3 Changes in NGAL with respect to development of renal failure (n = 20, no renal failure, 3 renal failure). All data are expressed as mean ± SD. RF indicates renal failure.
This is an important finding suggesting that mechanism of ballistic injury does not affect NGAL and, therefore, NGAL may have utility as an independent marker of AKI in the military intensive care setting. For reasons outlined above, it could be hypothesized that NGAL would rise with primary blast injury as the protein is expressed by both the gut and the lungs. This does not appear to be the case.
5.2. NGAL levels on admission to ITU and 12 and 24 hours later were significantly greater in those that subsequently died Neutrophil gelatinase–associated lipocalin levels rose to those associated with the development of renal failure [8],
Serum NGAL in ballistic injuries but in our series, renal failure was uncommon. Our findings suggest that in this group of fit, young patients, NGAL rise is related not just to the degree of renal insult but also perhaps the overall systemic inflammatory response.
5.3. NGAL did not rise immediately after injury In fact, it dropped significantly, possibly due to dilution, and only started to rise after arrival on the ITU. This finding is significant and conflicts with other investigators who have suggested NGAL rises within minutes [13] and may be useful as a triage tool in the emergency department [14]. The casualties in this series were all young and previously in very good health, and it may well be that there is a difference between the rapid renal insult of hypovolemia in trauma and the more insidious onset with sepsis or many of the disease states presenting to the emergency department. Our findings, with a rise in NGAL occurring after ICU admission, may reflect the release of NGAL as part of ischemia/reperfusion injury to the nephron or may simply represent the dilution of NGAL as shed blood is replaced with allogenic blood transfusion. In conclusion, although we consider our findings preliminary, an NGAL measurement in trauma cases on arrival at the ICU may prove to be a useful indicator not just of subsequent renal dysfunction but also of mortality. This possibility requires further investigation.
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