JBUR-4556; No. of Pages 8

Evaluation of haemoglobin in blister fluid as anindicator 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,*

aTissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of

Technology, Kelvin Grove, Brisbane, QLD, Australiab School of Biomedical Science, Faculty of Health, Queensland University of Technology, Kelvin Grove, Brisbane, QLD,

AustraliacCentre for Children’s Burns and Trauma Research, Queensland Children’s Medical Research Institute, Royal

Children’s Hospital, Herston, Brisbane, QLD, AustraliadWound Management Innovation Co-operative Research Centre, Kelvin Grove, Brisbane, QLD, Australia

b u r n s x x x ( 2 0 1 5 ) x x x – x x x

a r t i c l e i n f o

Article history:

Accepted 25 December 2014

Keywords:

Paediatric burns

Biomarkers

Haemoglobin

Wound depth

a b s t r a c t

The early and accurate assessment of burns is essential to inform patient treatment

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

adjunct diagnostic tool to assist experienced clinical judgement. Free circulating haemo-

globin has previously shown some promise as an indicator of burn depth in a murine animal

model. Using blister fluid collected from paediatric burn patients, haemoglobin abundance

was measured using semi-quantitative Western blot and immunoassays. Although a trend

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 fibri-

nous 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, KelvinGrove, Brisbane 4059, QLD, Australia. Tel.: +61 7 3138 6187; fax: +61 7 3138 6030.

E-mail addresses: catherine.tanzer@griffithuni.edu.au (C. Tanzer), dayle.sampson@qut.edu.au (D.L. Sampson),j2.broadbent@qut.edu.au (J.A. Broadbent), leila.cuttle@qut.edu.au (L. Cuttle), kempfm@mailbox.uq.edu.au (M. Kempf),royk@uq.edu.au (R.M. Kimble), z.upton@qut.edu.au (Z. Upton), a.parker@qut.edu.au (T.J. Parker).

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.elsevier.com/locate/burns

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

http://dx.doi.org/10.1016/j.burns.2014.12.0170305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.

JBUR-4556; No. of Pages 8

b u r n s x x x ( 2 0 1 5 ) x x x – x x x2

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

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

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 collec-

tion 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

375 mM tris(hydroxymethyl)aminomethane–hydrochloric

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/bisacry-

lamide (50:1) and 0.1% SDS in a total of 2 mL per gel.

Polymerisation was catalysed by addition of tetramethy-

lethylenediamine (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

n in blister fluid as an indicator of paediatric burn wound depth. Burns

b u r n s x x x ( 2 0 1 5 ) x x x – x x x 3

JBUR-4556; No. of Pages 8

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, Pen-

scola, 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 Saline-

Tween 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 anti-

body (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 normal-

ised 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 anti-

Human 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 3,305,50-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 spectrophotome-

ter (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/).

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

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 transfor-

mation was applied to the data prior to modelling. In this

analysis the additional clinical variables (independent vari-

ables) were explored to determine their influence on the

abundance of haemoglobin in burn wounds. These indepen-

dent variables included: patient age; gender; skin tone;

mechanism of injury; wound depth; wound location; percent-

age 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 spontane-

ous healing after injury. Importantly, in an effort to investigate

the wound environment and origin of the measured haemo-

globin, 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 immunoreac-

tive 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

n in blister fluid as an indicator of paediatric burn wound depth. Burns

Table 1 – Patient demographics and burn woundcharacteristics.

N

Patients 66

Samples 86

Median (range)

Age (months) 32.50 (6–189)

Total body surface

area burnt (%)

1 (0.1–50)

Days from injury to

sample collection

2 (0–18)

N (%)

Gender Male 42 (63.2%)

Skin complexion Light skin complexion 47 (71.2%)

Medium skin complexion 17 (25.8%)

Dark skin complexion 2 (3.0%)

Depth (by clinical

judgement)

Superficial partial thickness 46 (69.7%)

Deep partial thickness 15 (22.7%)

Full thickness 5 (7.6%)

Mechanism of injury Scald 28 (42.4%)

Contact 24 (36.4%)

Flame 11 (16.7%)

Other (e.g. radiation,

friction)

3 (4.6%)

Skin graft 10 (15.2%)

b u r n s x x x ( 2 0 1 5 ) x x x – x x x4

JBUR-4556; No. of Pages 8

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 concen-

tration 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

Fig. 1 – Haemoglobin abundance in blister fluid appeared to be associ

measured in 86 blister fluid samples using Western blot with d

point represents the mean of at least three replicates per samp

three: n = 61). Both bands (10 kDa and 30 kDa; inset) in each lan

data point represents the mean of three (n = 74) or two (n = 12)

mean of each depth group is depicted in grey, with one outlier ex

data sets a general trend of haemoglobin abundance decreasin

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

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 centri-

fuged samples contained detectable haemoglobin (Fig. 2B;

black) in concentrations comparable to un-centrifuged sam-

ples 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 immedi-

ately following centrifugation, as well as the removed cellular

debris. Significant differences were apparent in cell abun-

dance 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

ated with burn wound severity. Haemoglobin abundance was

ensitometry (A) and ELISA (B). (A) Each densitometry data

le W standard error of the mean (five: n = 3; four: n = 22; or

e were analysed and plotted independently. (B) Each ELISA

replicates per sample W standard error of the mean. The

cluded from the superficial/partial thickness group. In both

g with severity was observed, with a few notable outliers.

n in blister fluid as an indicator of paediatric burn wound depth. Burns

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.

b u r n s x x x ( 2 0 1 5 ) x x x – x x x 5

JBUR-4556; No. of Pages 8

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.

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

4. Discussion

A previous investigation has suggested that an increased

plasma concentration of free haemoglobin following a burn

n in blister fluid as an indicator of paediatric burn wound depth. Burns

Table 2 – Results from optimised LME model.

Coefficient F-value p-Value

Constant 0.0424 0.8376

Age (months) 2.9030 0.0940

Mechanism of injury 2.0500 0.0997

Days from injury to sample collection 25.280 0.0004*

Centrifugation at sampling 2.3155 0.1337

Days until spontaneous healing 5.4434 0.0396*

Site of injury 2.5554 0.0844

* Indicates p < 0.05.

b u r n s x x x ( 2 0 1 5 ) x x x – x x x6

JBUR-4556; No. of Pages 8

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 contami-

nation 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 haemo-

globin 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 erythro-

cytes [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 erythro-

cytes 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 preced-

ing vascular destruction) may occur locally at the wound site

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

and may not result from systemic alterations. Importantly,

this demonstrates the ability to measure site-specific altera-

tions 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 associa-

tion 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 recommenda-

tions [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 chemo-

tactic 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-epithelialisa-

tion. These relationships could be due to the destructive

wound microenvironment promoting damage among ery-

throcytes 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 microenviron-

ment. 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.

n in blister fluid as an indicator of paediatric burn wound depth. Burns

b u r n s x x x ( 2 0 1 5 ) x x x – x x x 7

JBUR-4556; No. of Pages 8

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.

r e f e r e n c e s

[1] Deitch EA, Wheelahan TM, Rose MP, Clothier J, Cotter J.Hypertrophic burn scars: analysis of variables. J Trauma1983;23:895–8.

[2] Monstrey S, Hoeksema H, Verbelen J, Pirayesh A, BlondeelP. Assessment of burn depth and burn wound healingpotential. Burns 2008;34:761–9.

[3] Prince HE. Biomarkers for diagnosing and monitoringautoimmune diseases. Biomarkers 2005;10 S1:S44–9(Biochemical Indicators of Exposure, Response, andSusceptibility to Chemicals).

[4] Fukuda I, et al. Potential plasma biomarkers for progressionof knee osteoarthritis using glycoproteomic analysiscoupled with a 2D-LC–MALDI system. Proteome Sci2012;10:36.

[5] Lin C-P, et al. Proteomic identification of plasmabiomarkers in uterine leiomyoma. Mol Biosyst 2012;8:1136–45.

[6] Zhang Z, et al. Three biomarkers identified from serumproteomic analysis for the detection of early stage ovariancancer. Cancer Res 2004;64:5882–90.

[7] Chen Y-T, et al. Multiplexed quantification of 63 proteins inhuman urine by multiple reaction monitoring-based massspectrometry for discovery of potential bladder cancerbiomarkers. J Proteomics 2012;75:3529–45.

[8] Li J, Zhang Z, Rosenzweig J, Wang YY, Chan DW. Proteomicsand bioinformatics approaches for identification of serumbiomarkers to detect breast cancer. Clin Chem2002;48:1296–304.

[9] Finnerty CC, et al. Determination of burn patient outcomeby large-scale quantitative discovery proteomics. Crit CareMed 2013;41:1421–34.

[10] Duke J, et al. An assessment of burn injury hospitalisationsof adolescents and young adults in Western Australia,1983–2008. Burns 2012;38:128–35.

[11] Wasiak J, et al. The epidemiology of burn injuries in anAustralian setting, 2000–2006. Burns 2009;35:1124–32.

[12] Eming SA, et al. Differential proteomic analysisdistinguishes tissue repair biomarker signatures in woundexudates obtained from normal healing and chronicwounds. J Proteome Res 2010;9:4758–66.

[13] Broszczak D, et al. Biochemical profiling of proteins andmetabolites in wound exudate from chronic woundenvironments. Wound Pract Res 2012;20:62–72.

[14] Fernandez ML, Broadbent JA, Shooter GK, Malda J, Upton Z.Development of an enhanced proteomic method to detectprognostic and diagnostic markers of healing in chronicwound fluid. Br J Dermatol 2008;158:281–90.

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

[15] Tarlton JF, et al. Prognostic value of markers of collagenremodeling in venous ulcers. Wound Repair Regen1999;7:347–55.

[16] Krisp C, et al. Proteome analysis reveals antiangiogenicenvironments in chronic wounds of diabetes mellitus type2 patients. Proteomics 2013;13:2670–81.

[17] Rayment EA, Upton Z, Shooter GK. Increased matrixmetalloproteinase-9 (MMP-9) activity observed in chronicwound fluid is related to the clinical severity of the ulcer. BrJ Dermatol 2008;158:951–61.

[18] Wong C-H, et al. Plasma free hemoglobin: a noveldiagnostic test for assessment of the depth of burn injury.Plast Reconstr Surg 2006;117:1206–13.

[19] Uchinuma E, Koganei Y, Shioya N, Yoshizato K. Biologicalevaluation of burn blister fluid. Ann Plast Surg 1988;20:225–30.

[20] Kool J, et al. Suction blister fluid as potential body fluid forbiomarker proteins. Proteomics 2007;7:3638–50.

[21] Widgerow AD, et al. The burn wound exudate—an under-utilized resource. Burns 2014;10. 1016/j.burns.2014.06.002.

[22] Jutkiewicz-Sypniewska J, Zembron-Lacny A, Puchała J,Szyszka K, Gajewski P. Oxidative stress in burnt children.Adv Med Sci 2006;51:316–20.

[23] Loebl EC, Baxter CR, Curreri PW. The mechanism oferythrocyte destruction in the early post-burn period. AnnSurg 1973;178:681–6.

[24] Till JR, Bruner GO, Ward LHPA. Thermal injury,intravascular hemolysis, and toxic oxygen products. J ClinInvest 1986;78:629–36.

[25] Brown NJ, Kimble RM, Gramotnev G, Rodger S, Cuttle L.Predictors of re-epithelialization in pediatric burn. Burns2014;40(4):751–8.

[26] Australian New Zealand Burn Association. Initialmanagement of small burns. Australian New Zealand BurnAssociation; 2014, Available from hwww.anzba.org.aui.

[27] Australian New Zealand Burn Association. Initialmanagement of severe burns. Australian New ZealandBurn Association; 2014, Available fromhwww.anzba.org.aui.

[28] Pan S-C, Wu L-W, Chen C-L, Shieh S-J, Chiu H-Y. Deeppartial thickness burn blister fluid promotesneovascularization in the early stage of burn woundhealing. Wound Repair Regen 2010;18:311–8.

[29] Pan S-C, Wu L-W, Chen C-L, Shieh S-J, Chiu H-Y.Angiogenin expression in burn blister fluid: implicationsfor its role in burn wound neovascularization. WoundRepair Regen 2012;20:731–9.

[30] Avniel S, et al. Involvement of the CXCL12/CXCR4 pathwayin the recovery of skin following burns. J Invest Dermatol2005;126:468–76.

[31] Mikhal’chik EV, et al. Comparative study of cytokinecontent in the plasma and wound exudate from childrenwith severe burns. Bull Exp Biol Med 2009;148:771–5.

[32] Inoue M, Zhou L-J, Gunji H, Ono I, Kaneko F. Effects ofcytokines in burn blister fluids on fibroblast proliferationand their inhibition with the use of neutralizing antibodies.Wound Repair Regen 1996;4:426–32.

[33] Prager MD, Baxter CR, Hartline B. Proteolytic activity inburn wound exudates and comparison of fibrin degradationproducts and protease inhibitors in exudates and sera. JBurn Care Rehabil 1994;15:130–6.

[34] Nissen NN, Gamelli RL, Polverini PJ, DiPietro LA. Differentialangiogenic and proliferative activity of surgical and burnwound fluids. J Trauma 2003;54:1205–10 (Discussion 1211).

[35] Ferrara JJ, Dyess DL, Luterman A, Curreri PW. Thesuppressive effect of subeschar tissue fluid upon in vitrocell-mediated immunologic function. J Burn Care Rehabil1988;9:584–8.

n in blister fluid as an indicator of paediatric burn wound depth. Burns

b u r n s x x x ( 2 0 1 5 ) x x x – x x x8

JBUR-4556; No. of Pages 8

[36] Dyess DL, Ferrara JJ, Luterman A, Curreri PW. Subeschartissue fluid: a source of cell-mediated immune suppressionin victims of severe thermal injury. J Burn Care Rehabil1991;12:101–5.

[37] Matuszczak E, Tylicka M, Debek W, Hermanowicz A,Ostrowska H. Correlation between circulating proteasome

Please cite this article in press as: Tanzer C, et al. Evaluation of haemoglobi(2015), http://dx.doi.org/10.1016/j.burns.2014.12.017

activity, total protein and c-reactive protein levelsfollowing burn in children. Burns 2014;40:842–7.

[38] Caulfield RH, Tyler MPH, Austyn JM, Dziewulski P,McGrouther DA. The relationship between protease/anti-protease profile, angiogenesis and re-epithelialisation inacute burn wounds. Burns 2008;34:474–86.

n in blister fluid as an indicator of paediatric burn wound depth. Burns