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Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth Catherine Tanzer a,b,c,d, Dayle L. Sampson a,b,d, James A. Broadbent a,b,d, Leila Cuttle a,b,c, Margit Kempf c, Roy M. Kimble c, Zee Upton a,b, Tony J. Parker a,b,* a Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Brisbane, QLD, Australia b School of Biomedical Science, Faculty of Health, Queensland University of Technology, Kelvin Grove, Brisbane, QLD, Australia c Centre for Children’s Burns and Trauma Research, Queensland Children’s Medical Research Institute, Royal Children’s Hospital, Herston, Brisbane, QLD, Australia d Wound Management Innovation Co-operative Research Centre, Kelvin Grove, Brisbane, QLD, Australia
article info
abstract
Article history:
The early and accurate assessment of burns is essential to inform patient treatment
Accepted 25 December 2014
regimens; however, this first critical step in clinical practice remains a challenge for specialist burns clinicians worldwide. In this regard, protein biomarkers are a potential
Keywords:
adjunct diagnostic tool to assist experienced clinical judgement. Free circulating haemo-
Paediatric burns
globin has previously shown some promise as an indicator of burn depth in a murine animal
Biomarkers
model. Using blister fluid collected from paediatric burn patients, haemoglobin abundance
Haemoglobin
was measured using semi-quantitative Western blot and immunoassays. Although a trend
Wound depth
was observed in which haemoglobin abundance increased with burn wound severity, several patient samples deviated significantly from this trend. Further, it was found that haemoglobin concentration decreased significantly when whole cells, cell debris and fibrinous matrix was removed from the blister fluid by centrifugation; although the relationship to depth was still present. Statistical analyses showed that haemoglobin abundance in the fluid was more strongly related to the time between injury and sample collection and the time taken for spontaneous re-epithelialisation. We hypothesise that prolonged exposure to the blister fluid microenvironment may result in an increased haemoglobin abundance due to erythrocyte lysis, and delayed wound healing. # 2015 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding author at: Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, Brisbane 4059, QLD, Australia. Tel.: +61 7 3138 6187; fax: +61 7 3138 6030. E-mail addresses: [email protected] (C. Tanzer), [email protected] (D.L. Sampson), [email protected] (J.A. Broadbent), [email protected] (L. Cuttle), [email protected] (M. Kempf), [email protected] (R.M. Kimble), [email protected] (Z. Upton), [email protected] (T.J. Parker). http://dx.doi.org/10.1016/j.burns.2014.12.017 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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1.
Introduction
The depth or severity of a burn is used by clinicians as the predominant variable to predict the time for spontaneous re-epithelialisation and the associated scarring outcomes [1]. Many methods and technologies have been developed to assist in measuring burn wound depth and healing potential; however, most of these are applied primarily in research as they have substantial clinical limitations, such as the size and cost of instruments or a requirement for specialist training [2]. Thus the gold standard of clinical care in paediatric burns is visual assessment by a clinician, which is both subjective and heavily dependent on the clinician’s training and experience. Therefore there remains the need for development of a more robust assessment tool. Protein biomarkers have been investigated as an indicator of the presence or progression of many common conditions, such as osteoarthritis, autoimmune diseases and several cancers [3–8]. To date few quantitative biological indicators or markers have been investigated for skin related conditions or specifically for burn wounds. Burn patient serum has been investigated for biomarkers predicting survival in severely burnt patients [9]; but as the incidence of burn mortality in Australia is relatively low, the majority of paediatric patients presenting to Australian burn centres do not face these survival concerns [10,11]. Studies focussing on biomarkers that could assist in predicting cutaneous wound healing trajectories have predominantly been conducted with the aim of assessing chronic non-healing wounds rather than acute wounds [12–15]. Although burn wound exudate has been used as a healing wound comparator in some chronic wound focussed studies [16,17], it is unclear whether a similar approach could be applied when assessing acute burn wounds only. Previously, the free circulating haemoglobin found in the plasma from a rat burn model has shown some promise as a biomarker of burn wound severity [18]; although this has not been further investigated in human patients. Moreover, the use of blood as a diagnostic sample is undesirable, particularly in the paediatric outpatient setting. In contrast to blood, blister fluid is readily available with minimal disruption to the patients or their medical treatment. As blister fluid is a plasma filtrate [19] proximal to the burn injury, there is potential for alterations in protein abundance which are detectable in blood to also be detectable in blister fluid. Blister fluid has previously been evaluated for its potential in biomarker discovery and measurement [20] and its utility in investigating the burn wound microenvironment [21], although it remains unclear whether changes in the local wound environment are detectable in this sample type or whether they would be masked by larger, systemic alterations. The ability to detect wound site-specific alterations in protein abundance may affect the ability of blister fluid to perform as a sample type for diagnostic or prognostic tests. This study therefore aimed to evaluate the use of haemoglobin as an indicator of burn wound severity in a population of paediatric burn patients using wound exudate. Analysis of a subset of samples from patients with multiple
burn sites was also conducted to determine whether site specific alterations in haemoglobin abundance could be detected.
2.
Methods
2.1.
Ethics statement
Ethical approval for this study was obtained from the Royal Children’s Hospital (RCH) Human Research Ethics Committee (No. HREC/11/QRCH/189) and the Queensland University of Technology Human Research Ethics Committee (QUT HREC Approval No. 1200000038). Clinical and demographic data from patients enrolled in the study were collected at the time of consent and at subsequent clinical visits.
2.2.
Sample collection and handling
Samples were collected through the Stuart Pegg Paediatric Burn Centre and the Department of Emergency Medicine at the RCH. During routine blister de-roofing procedures, fluid was acquired by either puncturing and aspirating the blister with a needle and syringe or puncturing the blister with scissors and collecting the fluid in a 200 mL ringcaps1 capillary pipette (Hirschmann Laborgerate, Eberstadt, Germany). To investigate free haemoglobin compared to that contained within erythrocytes, 28 samples collected by capillary pipette were centrifuged at 855 RCF immediately following collection to remove cells and debris. Prior to and immediately following centrifugation, representative samples were viewed using a Nikon Eclipse Ti inverted microscope or a Nikon Eclipse microscope, at 40 magnification and an aliquot of fluid was examined using a Neubauer chamber to perform erythrocyte counts. The pelleted cellular debris was stained with Giemsa and cell morphology was compared with a similarly stained whole blood sample. All samples were stored in aliquots at 80 8C. The total protein concentration of each sample was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, USA), as per the manufacturer’s instructions.
2.3.
SDS PAGE and Western blot
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) gels were cast using the Bio-Rad mini Protean system (Bio-Rad, Hercules, USA). The resolving gel contained tris(hydroxymethyl)aminomethane–hydrochloric 375 mM acid (Tris–HCl) pH 8.8, 10% acrylamide/bisacrylamide (50:1) and 0.1% SDS in a total of 4.5 mL per gel. The stacking gel contained 375 mM Tris–HCl pH 6.8, 4% acrylamide/bisacrylamide (50:1) and 0.1% SDS in a total of 2 mL per gel. Polymerisation was catalysed by addition of tetramethylethylenediamine (TEMED) and ammonium persulphate (APS). Samples (10 mg) and lysed human erythrocytes (1 mg; positive control) were prepared in NuPAGE lithium dodecyl sulphate sample buffer containing 100 mM dithiothreitol, incubated for 10 min at 70 8C and subject to electrophoresis at 180 V for 50 min in Tris–glycine SDS running buffer (25 mM Tris, 190 mM glycine, 0.1% SDS). Precision Plus protein
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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standard (Bio-Rad), served as a molecular weight indicator (250 kDa to 10 kDa). Electrophoretically resolved proteins were transferred onto a nitrocellulose blotting membrane (Pall Corporation, Penscola, USA) in Tris–glycine transfer buffer (25 mM Tris, 190 mM glycine and 20% ethanol) at 45 mA/gel on a Gibco semi-dry transfer apparatus (Life Technologies, Mulgrave, Australia). Membranes were blocked with 5% (w/v) bovine serum albumin fraction V (BSA; Life Technologies) in Tris Buffered SalineTween 20 (TBST), containing 100 mM Tris, 150 mM sodium chloride, pH 7.4 and 0.1% Tween 20, for 1 h at room temperature. Polyclonal goat anti-human haemoglobin antibody (R&D Systems, Minneapolis, USA) was diluted 1:10,000 in 0.5% BSA in TBST, prior to incubation with the membrane overnight at 4 8C. The membranes were washed with 1% BSA in TBST prior to incubation for 30 min at room temperature with Horse Radish Peroxidase (HRP) conjugated anti-goat IgG antibody (R&D Systems) diluted 1:20,000 in TBST. Following further washing the membranes were incubated with ECL Prime chemiluminescent substrate (GE Healthcare, Little Chalfont, UK) and detected with a Bio-Rad Gel Doc (Bio-Rad) and associated software. Densitometry was performed on the resulting images using ImageJ software (Version 1.47; http://imagej.nih.gov/ij). To minimise inter-blot variation, intensity readings were normalised to the intensity of the 10 kDa positive control band on each membrane.
2.4.
ELISA
Haemoglobin abundance in samples was measured using a Haemoglobin enzyme linked immunosorbent assay (ELISA) kit (ICL Labs, Portland, USA) following the manufacturer’s instructions. To ensure the readings fell within the linear range of the standard curve, samples containing high levels of haemoglobin, as detected by Western blot, were diluted 1:50,000 and all remaining samples were diluted 1:1000 in sample diluent. Haemoglobin standards, ranging from 200 ng/ mL to 6.25 ng/mL, and diluted samples were added in triplicate to wells of a 96 well plate pre-coated with affinity purified antiHuman haemoglobin antibodies. Following incubation, wells were washed and incubated with secondary anti-human haemoglobin antibodies conjugated with HRP. Following washes to remove unbound secondary antibody, wells were incubated in the presence of 3,30 5,50 -tetramethylbenzidine (TMB) solution and the reaction was stopped by addition of 300 mM sulphuric acid. The absorbance of samples was read at 450 nm using a Benchmark Plus microplate spectrophotometer (Bio-Rad) and the haemoglobin concentration of each sample was determined using the standard curve. Each sample was measured in triplicate assays.
2.5.
Data analysis
Analysis of patient demographics, univariate differences in haemoglobin abundance and plots, as measured by ELISA, were determined by Student’s t-test using GraphPad Prism (Version 6.03; www.graphpad.com). Multivariable models were produced using the ‘‘R’’ statistical computing and graphics program (Version 3.0.2; http://cran.r-project.org/).
3
The purpose of this analysis was to determine if levels of haemoglobin (the dependent variable) in burns patients was indicative of wound depth as others have demonstrated in a murine animal model [18]. Because the raw concentration of haemoglobin was not normally distributed, a log10 transformation was applied to the data prior to modelling. In this analysis the additional clinical variables (independent variables) were explored to determine their influence on the abundance of haemoglobin in burn wounds. These independent variables included: patient age; gender; skin tone; mechanism of injury; wound depth; wound location; percentage of total body surface area damaged; type and duration of initial first aid treatment; whether patients had undergone fluid resuscitation; wound grafted; and, days until spontaneous healing after injury. Importantly, in an effort to investigate the wound environment and origin of the measured haemoglobin, an additional categorical variable, ‘‘centrifugation at sampling’’, was added to each of the models to ensure it did not influence the level of detected haemoglobin. Each of these variables were considered as fixed effects in the models. Samples derived from wounds from different anatomical locations on the same patient were considered to be random effects during modelling. Due to the addition of random effects into the model, a linear mixed effects (LME) method was used. The model containing all of the above independent variables was optimised using a backward elimination process and compared using the Akaike information criterion score (AIC). Within the final model, clinical variables were considered to have a significant effect on the dependent variable (haemoglobin abundance) at p < 0.05.
3.
Results
For this study, 86 blister fluid samples were collected from 66 patients along with demographic and clinical data (Table 1). Patients were predominately male and of lighter skin complexion. The median age was 32 months (2 years 8 months), with a range of 6 months to 189 months (15 years 9 months). Burn wounds were predominately superficial partial thickness (as assessed by clinical judgement) and caused by scald. The median size of injury was 1% total body surface area (TBSA), with a range of 0.1% to 50% TBSA. Of the 86 samples, a subset of 28 samples was chosen for analysis from 14 patients who each contributed two samples collected at the same time point from separate blisters on disparate anatomical sites. This subset of 28 samples had similar demographic and burn characteristics to the total cohort although it contained a decreased proportion of patients with contact burns (7.14% compared to 36.36% in the total cohort) and no patients with full thickness burns. The overall relationship between haemoglobin abundance in blister fluid and burn severity was investigated using Western blot with subsequent densitometry (Fig. 1A) and ELISA (Fig. 1B). The Western blots revealed two immunoreactive bands at approximately 30 kDa and 10 kDa (Fig. 1A, inset). The 10 kDa band was present in more samples overall, while the 30 kDa band appeared predominately in samples with high intensity 10 kDa bands. In both the densitometry and ELISA data, a trend towards increased haemoglobin abundance with
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Table 1 – Patient demographics and burn wound characteristics. N Patients Samples Age (months) Total body surface area burnt (%) Days from injury to sample collection Male Light skin complexion Medium skin complexion Dark skin complexion Superficial partial thickness Depth (by clinical judgement) Deep partial thickness Full thickness Mechanism of injury Scald Contact Flame Other (e.g. radiation, friction) 10 (15.2%) Skin graft
Gender Skin complexion
66 86 Median (range) 32.50 (6–189) 1 (0.1–50) 2 (0–18) N (%) 42 (63.2%) 47 (71.2%) 17 (25.8%) 2 (3.0%) 46 (69.7%) 15 (22.7%) 5 (7.6%) 28 (42.4%) 24 (36.4%) 11 (16.7%) 3 (4.6%)
increased burn severity (burn depth and TBSA) was observed; however, notable outliers were also present. Specifically, some samples from small surface area superficial partial thickness burns were found to have comparable haemoglobin concentration to large surface area full thickness burns. In these instances, clinical confounders, such as potential needle stick injuries, were unable to account for the unexpectedly high haemoglobin concentrations. The site specific alterations in haemoglobin abundance were investigated by analysing the ELISA results for a subset of sample pairs which were collected at the same time point from separate blisters on different anatomical sites of the same
injury (Fig. 2A). Of the 28 samples, eight pairs contained detectable levels of haemoglobin and in six of these the haemoglobin concentration differed significantly between the pairs (all p < 0.05) suggesting a localised and non-systemic haemoglobin source. To investigate whether the detected haemoglobin was free in the blister fluid at the time of collection or contained within erythrocytes which subsequently lysed during storage, some samples were centrifuged at the time of collection to remove whole erythrocytes and other cellular debris. These centrifuged samples contained detectable haemoglobin (Fig. 2B; black) in concentrations comparable to un-centrifuged samples potentially containing lysed whole erythrocytes (Fig. 2B; grey). By centrifuging the samples to remove haemoglobin potentially originating from whole erythrocytes at the wound site, and measuring only haemoglobin free at the wound site, the trend remained similar, however the mean haemoglobin concentration decreased significantly ( p = 0.003). Further investigation into the impact of centrifugation of samples involved visualising samples prior to and immediately following centrifugation, as well as the removed cellular debris. Significant differences were apparent in cell abundance between samples prior (Fig. 3A; 976.0 RBCs/mL 762.1; range 0–4000) and subsequent (Fig. 3B; 10.0 RBCs/mL 7.07; range 0–30) to centrifugation ( p < 0.001). Staining of the removed pellet revealed blood and epithelial cells in addition to matrix-like structures (Fig. 3C). In an effort to determine potential correlation between the trend observed in the ELISA data and clinical factors, analysis of variables using LME methods was undertaken (Table 2). Of the covariates in the original model, all variables were removed after backward elimination except for patient age, mechanism of injury, days from injury to sample collection, days until spontaneous healing, centrifugation at sampling, and wound site. Of the covariates in the final model, days from injury to sample collection showed a strong significant effect ( p < 0.001). Based on this model, the haemoglobin levels
Fig. 1 – Haemoglobin abundance in blister fluid appeared to be associated with burn wound severity. Haemoglobin abundance was measured in 86 blister fluid samples using Western blot with densitometry (A) and ELISA (B). (A) Each densitometry data point represents the mean of at least three replicates per sample W standard error of the mean (five: n = 3; four: n = 22; or three: n = 61). Both bands (10 kDa and 30 kDa; inset) in each lane were analysed and plotted independently. (B) Each ELISA data point represents the mean of three (n = 74) or two (n = 12) replicates per sample W standard error of the mean. The mean of each depth group is depicted in grey, with one outlier excluded from the superficial/partial thickness group. In both data sets a general trend of haemoglobin abundance decreasing with severity was observed, with a few notable outliers. Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Fig. 2 – Sample collection and processing techniques affect the haemoglobin concentration of blister fluid. (A) Fourteen pairs of samples were collected from separate blisters at the same sampling time point and subjected to ELISA assay. Haemoglobin concentrations differed significantly between sampling sites in six of the eight patients with detectable haemoglobin levels (* p < 0.05; ** p < 0.01). (B) Samples were either stored immediately after collection at S80 8C (black) or centrifuged to remove cell debris prior to storage (grey) and subsequently subjected to ELISA assay. Each data point represents the mean of three (n = 74) or two (n = 12) replicates W standard error of the mean. The mean haemoglobin abundance (full line and dashed line, respectively) differed significantly between the two groups (236,237 ng/mL versus 3832 ng/mL; p = 0.003).
Fig. 3 – Centrifugation removes the majority of erythrocytes and a molecular matrix from blister fluid samples. Representative blister fluid samples were viewed prior to (A) and immediately following (B) centrifugation. A significant reduction in erythrocyte counts was observed between groups (976.0 RBCs/mL W 762.1 versus 10.0 RBCs/mL W 7.07; p < 0.001). Giemsa staining and microscopy of the cellular debris (C) removed by centrifugation revealed the presence of erythrocytes, epithelial cells and a molecular matrix. Intact erythrocyte size and morphology was compared with a whole blood sample (D) serving as positive control; scale bar = 50 mm.
increased by log10(0.3) ng/mL per day following the burn injury. Time to spontaneous healing after injury was also significant ( p < 0.05), indicating that the haemoglobin level increased by log10(0.02) ng/mL for every day that passed until spontaneous healing occurs.
4.
Discussion
A previous investigation has suggested that an increased plasma concentration of free haemoglobin following a burn
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Table 2 – Results from optimised LME model. Coefficient
F-value
p-Value
Constant Age (months) Mechanism of injury Days from injury to sample collection Centrifugation at sampling Days until spontaneous healing Site of injury
0.0424 2.9030 2.0500 25.280 2.3155 5.4434 2.5554
0.8376 0.0940 0.0997 0.0004* 0.1337 0.0396* 0.0844
*
Indicates p < 0.05.
injury may be associated with the severity of the burn [18]. The study described herein sought to investigate this hypothesis further in a clinical population using Western blot and ELISA techniques. These techniques differ from the optical methods employed in the original study to allow a completely independent and complementary investigation. Although both Western blot and ELISA resulted in the observation of similar trends, the ELISA enabled quantitative measurement of the protein. In both testing methods, there appeared to be a trend towards increased haemoglobin levels in blister fluid of patients with severe burns; however, several patient samples, both deep partial and superficial partial thickness burns, were observed to deviate significantly from this trend. While needle stick injury is the most likely cause of haemoglobin contamination and has the potential to explain the significant levels of haemoglobin observed in some of the least severe wound samples, the sampling methods used for this study were designed to reduce this risk and any potential needle stick samples were excluded. While it has been suggested that circulating free haemoglobin may be correlated to burn severity [18], there is also increasing evidence that reactive oxygen species are released into the blood following a burn injury [22]. This potentially increases the fragility and decreases the half-life of erythrocytes [23,24]. Thus, increased fragility of erythrocytes in patients with thermal injury to the deep dermis would potentially lead to increased erythrocyte lysis and the release of free haemoglobin into the wound, among other cellular components. By analysing samples with and without whole cells, cellular debris and fibrinous matrix, we were able to determine whether free haemoglobin or that contained within whole erythrocytes at sampling is responsible for the observed haemoglobin abundance. Our results demonstrated that, while haemoglobin is able to be detected following the removal of the majority of cells and cellular debris, the concentration is observed to be significantly lower. From this data, we postulate that thermal injury creates a critical level of damage to the dermal vasculature such that whole erythrocytes enter the burn wound. In combination with the increased fragility of circulating erythrocytes, both free haemoglobin and that contained within erythrocytes are increased substantially. Significant differences in haemoglobin concentration were observed in samples collected from different blisters from the same individual patients. These data suggest that the alteration in haemoglobin abundance (and postulated preceding vascular destruction) may occur locally at the wound site
and may not result from systemic alterations. Importantly, this demonstrates the ability to measure site-specific alterations in haemoglobin. Burns are rarely of uniform depth and thus the ability to discriminate the deeper areas from more superficial areas is crucial. Multivariable analyses identified a significant relationship between haemoglobin abundance and both time from injury to sampling and time to spontaneous re-epithelialisation. In most cases, a delay in sample collection was due to delayed presentation to the treating tertiary burn centre. An association has previously been reported between the number of days taken to present to the burn centre and time to spontaneous re-epithelialisation [25]. In that study, it was hypothesised that the delayed healing observed within patients with delayed presentation could be due to the delay in access to specialised treatment from an expert burn team or delayed removal of devitalised tissue in the injured area [25]. While much debate surrounds the practise of prompt blister de-roofing and removal of unviable tissue, this is standard protocol in the Stuart Pegg Paediatric Burn Centre, in keeping with the Australian and New Zealand Burn Association recommendations [26,27]. This measure is thought to reduce the risk of infection associated with uncontrolled blister rupture and the prolonged presence of devitalised epithelium. Previous work has suggested that cytokines, angiogenic factors and chemotactic factors present in blister fluid may assist healing in the initial phases [28–32]; however, other authors suggest that the prolonged exposure to proteolytic and immunosuppressive factors within the fluid [33–37] may delay healing and lead to scarring. While these studies vary in their methodology and use of controls, the overall results suggest that a balance between these factors within the wound environment may be integral for regulating normal healing [38]. Although haemoglobin abundance has shown promise as a potential biomarker of burn wound severity in a murine animal model, this study has highlighted many factors which limit the robustness and accuracy of its measurement for clinical use. Several samples significantly deviated from a linear trend and measurements have been shown to vary based on the site of collection on the same patient and sample processing techniques. Of biological interest is the hypothesis that thermal injury of any severity causes enough damage to the dermal vasculature to allow whole red blood cells to enter the wound site. The results also showed a more significant relationship between haemoglobin abundance and both time from injury to sample collection and time to re-epithelialisation. These relationships could be due to the destructive wound microenvironment promoting damage among erythrocytes and migrating skin cells. Looking toward the future, additional research might focus on the effect of free haemoglobin and the diffuse matrix observed within blister fluid or the measurement of reactive oxygen species for their potential biological effects within the wound microenvironment. Moreover, there needs to be a concerted effort to identify quantitative clinical markers to assist clinical decision making thereby decreasing hospital costs and improving patient outcomes. It is likely that a panel of biomarkers covering the inflammatory and angiogenic status of wound fluid will be required to accurately assess the wound environment and enable early prediction of healing outcomes.
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Until such time as a prognostic panel of biomarkers is developed, early accurate assessment of burns will remain a challenge to clinicians.
Acknowledgements The authors would like to thank the patients and families who participated in this study and the staff of the Stuart Pegg Paediatric Burn Centre for their assistance during patient recruitment and sample collection. This study was supported by the Wound Management Innovation Co-operative Research Centre and the Children’s Health Foundation.
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Available online at www.sciencedirect.com
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Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth Catherine Tanzer a,b,c,d, Dayle L. Sampson a,b,d, James A. Broadbent a,b,d, Leila Cuttle a,b,c, Margit Kempf c, Roy M. Kimble c, Zee Upton a,b, Tony J. Parker a,b,* a Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Brisbane, QLD, Australia b School of Biomedical Science, Faculty of Health, Queensland University of Technology, Kelvin Grove, Brisbane, QLD, Australia c Centre for Children’s Burns and Trauma Research, Queensland Children’s Medical Research Institute, Royal Children’s Hospital, Herston, Brisbane, QLD, Australia d Wound Management Innovation Co-operative Research Centre, Kelvin Grove, Brisbane, QLD, Australia
article info
abstract
Article history:
The early and accurate assessment of burns is essential to inform patient treatment
Accepted 25 December 2014
regimens; however, this first critical step in clinical practice remains a challenge for specialist burns clinicians worldwide. In this regard, protein biomarkers are a potential
Keywords:
adjunct diagnostic tool to assist experienced clinical judgement. Free circulating haemo-
Paediatric burns
globin has previously shown some promise as an indicator of burn depth in a murine animal
Biomarkers
model. Using blister fluid collected from paediatric burn patients, haemoglobin abundance
Haemoglobin
was measured using semi-quantitative Western blot and immunoassays. Although a trend
Wound depth
was observed in which haemoglobin abundance increased with burn wound severity, several patient samples deviated significantly from this trend. Further, it was found that haemoglobin concentration decreased significantly when whole cells, cell debris and fibrinous matrix was removed from the blister fluid by centrifugation; although the relationship to depth was still present. Statistical analyses showed that haemoglobin abundance in the fluid was more strongly related to the time between injury and sample collection and the time taken for spontaneous re-epithelialisation. We hypothesise that prolonged exposure to the blister fluid microenvironment may result in an increased haemoglobin abundance due to erythrocyte lysis, and delayed wound healing. # 2015 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding author at: Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, Brisbane 4059, QLD, Australia. Tel.: +61 7 3138 6187; fax: +61 7 3138 6030. E-mail addresses: [email protected] (C. Tanzer), [email protected] (D.L. Sampson), [email protected] (J.A. Broadbent), [email protected] (L. Cuttle), [email protected] (M. Kempf), [email protected] (R.M. Kimble), [email protected] (Z. Upton), [email protected] (T.J. Parker). http://dx.doi.org/10.1016/j.burns.2014.12.017 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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1.
Introduction
The depth or severity of a burn is used by clinicians as the predominant variable to predict the time for spontaneous re-epithelialisation and the associated scarring outcomes [1]. Many methods and technologies have been developed to assist in measuring burn wound depth and healing potential; however, most of these are applied primarily in research as they have substantial clinical limitations, such as the size and cost of instruments or a requirement for specialist training [2]. Thus the gold standard of clinical care in paediatric burns is visual assessment by a clinician, which is both subjective and heavily dependent on the clinician’s training and experience. Therefore there remains the need for development of a more robust assessment tool. Protein biomarkers have been investigated as an indicator of the presence or progression of many common conditions, such as osteoarthritis, autoimmune diseases and several cancers [3–8]. To date few quantitative biological indicators or markers have been investigated for skin related conditions or specifically for burn wounds. Burn patient serum has been investigated for biomarkers predicting survival in severely burnt patients [9]; but as the incidence of burn mortality in Australia is relatively low, the majority of paediatric patients presenting to Australian burn centres do not face these survival concerns [10,11]. Studies focussing on biomarkers that could assist in predicting cutaneous wound healing trajectories have predominantly been conducted with the aim of assessing chronic non-healing wounds rather than acute wounds [12–15]. Although burn wound exudate has been used as a healing wound comparator in some chronic wound focussed studies [16,17], it is unclear whether a similar approach could be applied when assessing acute burn wounds only. Previously, the free circulating haemoglobin found in the plasma from a rat burn model has shown some promise as a biomarker of burn wound severity [18]; although this has not been further investigated in human patients. Moreover, the use of blood as a diagnostic sample is undesirable, particularly in the paediatric outpatient setting. In contrast to blood, blister fluid is readily available with minimal disruption to the patients or their medical treatment. As blister fluid is a plasma filtrate [19] proximal to the burn injury, there is potential for alterations in protein abundance which are detectable in blood to also be detectable in blister fluid. Blister fluid has previously been evaluated for its potential in biomarker discovery and measurement [20] and its utility in investigating the burn wound microenvironment [21], although it remains unclear whether changes in the local wound environment are detectable in this sample type or whether they would be masked by larger, systemic alterations. The ability to detect wound site-specific alterations in protein abundance may affect the ability of blister fluid to perform as a sample type for diagnostic or prognostic tests. This study therefore aimed to evaluate the use of haemoglobin as an indicator of burn wound severity in a population of paediatric burn patients using wound exudate. Analysis of a subset of samples from patients with multiple
burn sites was also conducted to determine whether site specific alterations in haemoglobin abundance could be detected.
2.
Methods
2.1.
Ethics statement
Ethical approval for this study was obtained from the Royal Children’s Hospital (RCH) Human Research Ethics Committee (No. HREC/11/QRCH/189) and the Queensland University of Technology Human Research Ethics Committee (QUT HREC Approval No. 1200000038). Clinical and demographic data from patients enrolled in the study were collected at the time of consent and at subsequent clinical visits.
2.2.
Sample collection and handling
Samples were collected through the Stuart Pegg Paediatric Burn Centre and the Department of Emergency Medicine at the RCH. During routine blister de-roofing procedures, fluid was acquired by either puncturing and aspirating the blister with a needle and syringe or puncturing the blister with scissors and collecting the fluid in a 200 mL ringcaps1 capillary pipette (Hirschmann Laborgerate, Eberstadt, Germany). To investigate free haemoglobin compared to that contained within erythrocytes, 28 samples collected by capillary pipette were centrifuged at 855 RCF immediately following collection to remove cells and debris. Prior to and immediately following centrifugation, representative samples were viewed using a Nikon Eclipse Ti inverted microscope or a Nikon Eclipse microscope, at 40 magnification and an aliquot of fluid was examined using a Neubauer chamber to perform erythrocyte counts. The pelleted cellular debris was stained with Giemsa and cell morphology was compared with a similarly stained whole blood sample. All samples were stored in aliquots at 80 8C. The total protein concentration of each sample was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, USA), as per the manufacturer’s instructions.
2.3.
SDS PAGE and Western blot
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) gels were cast using the Bio-Rad mini Protean system (Bio-Rad, Hercules, USA). The resolving gel contained tris(hydroxymethyl)aminomethane–hydrochloric 375 mM acid (Tris–HCl) pH 8.8, 10% acrylamide/bisacrylamide (50:1) and 0.1% SDS in a total of 4.5 mL per gel. The stacking gel contained 375 mM Tris–HCl pH 6.8, 4% acrylamide/bisacrylamide (50:1) and 0.1% SDS in a total of 2 mL per gel. Polymerisation was catalysed by addition of tetramethylethylenediamine (TEMED) and ammonium persulphate (APS). Samples (10 mg) and lysed human erythrocytes (1 mg; positive control) were prepared in NuPAGE lithium dodecyl sulphate sample buffer containing 100 mM dithiothreitol, incubated for 10 min at 70 8C and subject to electrophoresis at 180 V for 50 min in Tris–glycine SDS running buffer (25 mM Tris, 190 mM glycine, 0.1% SDS). Precision Plus protein
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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standard (Bio-Rad), served as a molecular weight indicator (250 kDa to 10 kDa). Electrophoretically resolved proteins were transferred onto a nitrocellulose blotting membrane (Pall Corporation, Penscola, USA) in Tris–glycine transfer buffer (25 mM Tris, 190 mM glycine and 20% ethanol) at 45 mA/gel on a Gibco semi-dry transfer apparatus (Life Technologies, Mulgrave, Australia). Membranes were blocked with 5% (w/v) bovine serum albumin fraction V (BSA; Life Technologies) in Tris Buffered SalineTween 20 (TBST), containing 100 mM Tris, 150 mM sodium chloride, pH 7.4 and 0.1% Tween 20, for 1 h at room temperature. Polyclonal goat anti-human haemoglobin antibody (R&D Systems, Minneapolis, USA) was diluted 1:10,000 in 0.5% BSA in TBST, prior to incubation with the membrane overnight at 4 8C. The membranes were washed with 1% BSA in TBST prior to incubation for 30 min at room temperature with Horse Radish Peroxidase (HRP) conjugated anti-goat IgG antibody (R&D Systems) diluted 1:20,000 in TBST. Following further washing the membranes were incubated with ECL Prime chemiluminescent substrate (GE Healthcare, Little Chalfont, UK) and detected with a Bio-Rad Gel Doc (Bio-Rad) and associated software. Densitometry was performed on the resulting images using ImageJ software (Version 1.47; http://imagej.nih.gov/ij). To minimise inter-blot variation, intensity readings were normalised to the intensity of the 10 kDa positive control band on each membrane.
2.4.
ELISA
Haemoglobin abundance in samples was measured using a Haemoglobin enzyme linked immunosorbent assay (ELISA) kit (ICL Labs, Portland, USA) following the manufacturer’s instructions. To ensure the readings fell within the linear range of the standard curve, samples containing high levels of haemoglobin, as detected by Western blot, were diluted 1:50,000 and all remaining samples were diluted 1:1000 in sample diluent. Haemoglobin standards, ranging from 200 ng/ mL to 6.25 ng/mL, and diluted samples were added in triplicate to wells of a 96 well plate pre-coated with affinity purified antiHuman haemoglobin antibodies. Following incubation, wells were washed and incubated with secondary anti-human haemoglobin antibodies conjugated with HRP. Following washes to remove unbound secondary antibody, wells were incubated in the presence of 3,30 5,50 -tetramethylbenzidine (TMB) solution and the reaction was stopped by addition of 300 mM sulphuric acid. The absorbance of samples was read at 450 nm using a Benchmark Plus microplate spectrophotometer (Bio-Rad) and the haemoglobin concentration of each sample was determined using the standard curve. Each sample was measured in triplicate assays.
2.5.
Data analysis
Analysis of patient demographics, univariate differences in haemoglobin abundance and plots, as measured by ELISA, were determined by Student’s t-test using GraphPad Prism (Version 6.03; www.graphpad.com). Multivariable models were produced using the ‘‘R’’ statistical computing and graphics program (Version 3.0.2; http://cran.r-project.org/).
3
The purpose of this analysis was to determine if levels of haemoglobin (the dependent variable) in burns patients was indicative of wound depth as others have demonstrated in a murine animal model [18]. Because the raw concentration of haemoglobin was not normally distributed, a log10 transformation was applied to the data prior to modelling. In this analysis the additional clinical variables (independent variables) were explored to determine their influence on the abundance of haemoglobin in burn wounds. These independent variables included: patient age; gender; skin tone; mechanism of injury; wound depth; wound location; percentage of total body surface area damaged; type and duration of initial first aid treatment; whether patients had undergone fluid resuscitation; wound grafted; and, days until spontaneous healing after injury. Importantly, in an effort to investigate the wound environment and origin of the measured haemoglobin, an additional categorical variable, ‘‘centrifugation at sampling’’, was added to each of the models to ensure it did not influence the level of detected haemoglobin. Each of these variables were considered as fixed effects in the models. Samples derived from wounds from different anatomical locations on the same patient were considered to be random effects during modelling. Due to the addition of random effects into the model, a linear mixed effects (LME) method was used. The model containing all of the above independent variables was optimised using a backward elimination process and compared using the Akaike information criterion score (AIC). Within the final model, clinical variables were considered to have a significant effect on the dependent variable (haemoglobin abundance) at p < 0.05.
3.
Results
For this study, 86 blister fluid samples were collected from 66 patients along with demographic and clinical data (Table 1). Patients were predominately male and of lighter skin complexion. The median age was 32 months (2 years 8 months), with a range of 6 months to 189 months (15 years 9 months). Burn wounds were predominately superficial partial thickness (as assessed by clinical judgement) and caused by scald. The median size of injury was 1% total body surface area (TBSA), with a range of 0.1% to 50% TBSA. Of the 86 samples, a subset of 28 samples was chosen for analysis from 14 patients who each contributed two samples collected at the same time point from separate blisters on disparate anatomical sites. This subset of 28 samples had similar demographic and burn characteristics to the total cohort although it contained a decreased proportion of patients with contact burns (7.14% compared to 36.36% in the total cohort) and no patients with full thickness burns. The overall relationship between haemoglobin abundance in blister fluid and burn severity was investigated using Western blot with subsequent densitometry (Fig. 1A) and ELISA (Fig. 1B). The Western blots revealed two immunoreactive bands at approximately 30 kDa and 10 kDa (Fig. 1A, inset). The 10 kDa band was present in more samples overall, while the 30 kDa band appeared predominately in samples with high intensity 10 kDa bands. In both the densitometry and ELISA data, a trend towards increased haemoglobin abundance with
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Table 1 – Patient demographics and burn wound characteristics. N Patients Samples Age (months) Total body surface area burnt (%) Days from injury to sample collection Male Light skin complexion Medium skin complexion Dark skin complexion Superficial partial thickness Depth (by clinical judgement) Deep partial thickness Full thickness Mechanism of injury Scald Contact Flame Other (e.g. radiation, friction) 10 (15.2%) Skin graft
Gender Skin complexion
66 86 Median (range) 32.50 (6–189) 1 (0.1–50) 2 (0–18) N (%) 42 (63.2%) 47 (71.2%) 17 (25.8%) 2 (3.0%) 46 (69.7%) 15 (22.7%) 5 (7.6%) 28 (42.4%) 24 (36.4%) 11 (16.7%) 3 (4.6%)
increased burn severity (burn depth and TBSA) was observed; however, notable outliers were also present. Specifically, some samples from small surface area superficial partial thickness burns were found to have comparable haemoglobin concentration to large surface area full thickness burns. In these instances, clinical confounders, such as potential needle stick injuries, were unable to account for the unexpectedly high haemoglobin concentrations. The site specific alterations in haemoglobin abundance were investigated by analysing the ELISA results for a subset of sample pairs which were collected at the same time point from separate blisters on different anatomical sites of the same
injury (Fig. 2A). Of the 28 samples, eight pairs contained detectable levels of haemoglobin and in six of these the haemoglobin concentration differed significantly between the pairs (all p < 0.05) suggesting a localised and non-systemic haemoglobin source. To investigate whether the detected haemoglobin was free in the blister fluid at the time of collection or contained within erythrocytes which subsequently lysed during storage, some samples were centrifuged at the time of collection to remove whole erythrocytes and other cellular debris. These centrifuged samples contained detectable haemoglobin (Fig. 2B; black) in concentrations comparable to un-centrifuged samples potentially containing lysed whole erythrocytes (Fig. 2B; grey). By centrifuging the samples to remove haemoglobin potentially originating from whole erythrocytes at the wound site, and measuring only haemoglobin free at the wound site, the trend remained similar, however the mean haemoglobin concentration decreased significantly ( p = 0.003). Further investigation into the impact of centrifugation of samples involved visualising samples prior to and immediately following centrifugation, as well as the removed cellular debris. Significant differences were apparent in cell abundance between samples prior (Fig. 3A; 976.0 RBCs/mL 762.1; range 0–4000) and subsequent (Fig. 3B; 10.0 RBCs/mL 7.07; range 0–30) to centrifugation ( p < 0.001). Staining of the removed pellet revealed blood and epithelial cells in addition to matrix-like structures (Fig. 3C). In an effort to determine potential correlation between the trend observed in the ELISA data and clinical factors, analysis of variables using LME methods was undertaken (Table 2). Of the covariates in the original model, all variables were removed after backward elimination except for patient age, mechanism of injury, days from injury to sample collection, days until spontaneous healing, centrifugation at sampling, and wound site. Of the covariates in the final model, days from injury to sample collection showed a strong significant effect ( p < 0.001). Based on this model, the haemoglobin levels
Fig. 1 – Haemoglobin abundance in blister fluid appeared to be associated with burn wound severity. Haemoglobin abundance was measured in 86 blister fluid samples using Western blot with densitometry (A) and ELISA (B). (A) Each densitometry data point represents the mean of at least three replicates per sample W standard error of the mean (five: n = 3; four: n = 22; or three: n = 61). Both bands (10 kDa and 30 kDa; inset) in each lane were analysed and plotted independently. (B) Each ELISA data point represents the mean of three (n = 74) or two (n = 12) replicates per sample W standard error of the mean. The mean of each depth group is depicted in grey, with one outlier excluded from the superficial/partial thickness group. In both data sets a general trend of haemoglobin abundance decreasing with severity was observed, with a few notable outliers. Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Fig. 2 – Sample collection and processing techniques affect the haemoglobin concentration of blister fluid. (A) Fourteen pairs of samples were collected from separate blisters at the same sampling time point and subjected to ELISA assay. Haemoglobin concentrations differed significantly between sampling sites in six of the eight patients with detectable haemoglobin levels (* p < 0.05; ** p < 0.01). (B) Samples were either stored immediately after collection at S80 8C (black) or centrifuged to remove cell debris prior to storage (grey) and subsequently subjected to ELISA assay. Each data point represents the mean of three (n = 74) or two (n = 12) replicates W standard error of the mean. The mean haemoglobin abundance (full line and dashed line, respectively) differed significantly between the two groups (236,237 ng/mL versus 3832 ng/mL; p = 0.003).
Fig. 3 – Centrifugation removes the majority of erythrocytes and a molecular matrix from blister fluid samples. Representative blister fluid samples were viewed prior to (A) and immediately following (B) centrifugation. A significant reduction in erythrocyte counts was observed between groups (976.0 RBCs/mL W 762.1 versus 10.0 RBCs/mL W 7.07; p < 0.001). Giemsa staining and microscopy of the cellular debris (C) removed by centrifugation revealed the presence of erythrocytes, epithelial cells and a molecular matrix. Intact erythrocyte size and morphology was compared with a whole blood sample (D) serving as positive control; scale bar = 50 mm.
increased by log10(0.3) ng/mL per day following the burn injury. Time to spontaneous healing after injury was also significant ( p < 0.05), indicating that the haemoglobin level increased by log10(0.02) ng/mL for every day that passed until spontaneous healing occurs.
4.
Discussion
A previous investigation has suggested that an increased plasma concentration of free haemoglobin following a burn
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Table 2 – Results from optimised LME model. Coefficient
F-value
p-Value
Constant Age (months) Mechanism of injury Days from injury to sample collection Centrifugation at sampling Days until spontaneous healing Site of injury
0.0424 2.9030 2.0500 25.280 2.3155 5.4434 2.5554
0.8376 0.0940 0.0997 0.0004* 0.1337 0.0396* 0.0844
*
Indicates p < 0.05.
injury may be associated with the severity of the burn [18]. The study described herein sought to investigate this hypothesis further in a clinical population using Western blot and ELISA techniques. These techniques differ from the optical methods employed in the original study to allow a completely independent and complementary investigation. Although both Western blot and ELISA resulted in the observation of similar trends, the ELISA enabled quantitative measurement of the protein. In both testing methods, there appeared to be a trend towards increased haemoglobin levels in blister fluid of patients with severe burns; however, several patient samples, both deep partial and superficial partial thickness burns, were observed to deviate significantly from this trend. While needle stick injury is the most likely cause of haemoglobin contamination and has the potential to explain the significant levels of haemoglobin observed in some of the least severe wound samples, the sampling methods used for this study were designed to reduce this risk and any potential needle stick samples were excluded. While it has been suggested that circulating free haemoglobin may be correlated to burn severity [18], there is also increasing evidence that reactive oxygen species are released into the blood following a burn injury [22]. This potentially increases the fragility and decreases the half-life of erythrocytes [23,24]. Thus, increased fragility of erythrocytes in patients with thermal injury to the deep dermis would potentially lead to increased erythrocyte lysis and the release of free haemoglobin into the wound, among other cellular components. By analysing samples with and without whole cells, cellular debris and fibrinous matrix, we were able to determine whether free haemoglobin or that contained within whole erythrocytes at sampling is responsible for the observed haemoglobin abundance. Our results demonstrated that, while haemoglobin is able to be detected following the removal of the majority of cells and cellular debris, the concentration is observed to be significantly lower. From this data, we postulate that thermal injury creates a critical level of damage to the dermal vasculature such that whole erythrocytes enter the burn wound. In combination with the increased fragility of circulating erythrocytes, both free haemoglobin and that contained within erythrocytes are increased substantially. Significant differences in haemoglobin concentration were observed in samples collected from different blisters from the same individual patients. These data suggest that the alteration in haemoglobin abundance (and postulated preceding vascular destruction) may occur locally at the wound site
and may not result from systemic alterations. Importantly, this demonstrates the ability to measure site-specific alterations in haemoglobin. Burns are rarely of uniform depth and thus the ability to discriminate the deeper areas from more superficial areas is crucial. Multivariable analyses identified a significant relationship between haemoglobin abundance and both time from injury to sampling and time to spontaneous re-epithelialisation. In most cases, a delay in sample collection was due to delayed presentation to the treating tertiary burn centre. An association has previously been reported between the number of days taken to present to the burn centre and time to spontaneous re-epithelialisation [25]. In that study, it was hypothesised that the delayed healing observed within patients with delayed presentation could be due to the delay in access to specialised treatment from an expert burn team or delayed removal of devitalised tissue in the injured area [25]. While much debate surrounds the practise of prompt blister de-roofing and removal of unviable tissue, this is standard protocol in the Stuart Pegg Paediatric Burn Centre, in keeping with the Australian and New Zealand Burn Association recommendations [26,27]. This measure is thought to reduce the risk of infection associated with uncontrolled blister rupture and the prolonged presence of devitalised epithelium. Previous work has suggested that cytokines, angiogenic factors and chemotactic factors present in blister fluid may assist healing in the initial phases [28–32]; however, other authors suggest that the prolonged exposure to proteolytic and immunosuppressive factors within the fluid [33–37] may delay healing and lead to scarring. While these studies vary in their methodology and use of controls, the overall results suggest that a balance between these factors within the wound environment may be integral for regulating normal healing [38]. Although haemoglobin abundance has shown promise as a potential biomarker of burn wound severity in a murine animal model, this study has highlighted many factors which limit the robustness and accuracy of its measurement for clinical use. Several samples significantly deviated from a linear trend and measurements have been shown to vary based on the site of collection on the same patient and sample processing techniques. Of biological interest is the hypothesis that thermal injury of any severity causes enough damage to the dermal vasculature to allow whole red blood cells to enter the wound site. The results also showed a more significant relationship between haemoglobin abundance and both time from injury to sample collection and time to re-epithelialisation. These relationships could be due to the destructive wound microenvironment promoting damage among erythrocytes and migrating skin cells. Looking toward the future, additional research might focus on the effect of free haemoglobin and the diffuse matrix observed within blister fluid or the measurement of reactive oxygen species for their potential biological effects within the wound microenvironment. Moreover, there needs to be a concerted effort to identify quantitative clinical markers to assist clinical decision making thereby decreasing hospital costs and improving patient outcomes. It is likely that a panel of biomarkers covering the inflammatory and angiogenic status of wound fluid will be required to accurately assess the wound environment and enable early prediction of healing outcomes.
Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.017
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Until such time as a prognostic panel of biomarkers is developed, early accurate assessment of burns will remain a challenge to clinicians.
Acknowledgements The authors would like to thank the patients and families who participated in this study and the staff of the Stuart Pegg Paediatric Burn Centre for their assistance during patient recruitment and sample collection. This study was supported by the Wound Management Innovation Co-operative Research Centre and the Children’s Health Foundation.
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