1

Evaluation of haemoglobin in blister fluid as an indicator of paediatric

burn wound depth

Abstract

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 haemoglobin 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 semiquantitative 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 reepithelialisation. 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.

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 clinicians
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 [38]. To date few quantitative biological indicators or
markers have been investigated for skin related conditions or specifically for burn

2
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 [1215]. 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 Childrens 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
manufacturers 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)aminomethanehydrochloric acid (TrisHCl) 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 TrisHCl 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 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 Trisglycine SDS running buffer (25 mM Tris,
190 mM glycine, 0.1% SDS). Precision Plus protein 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 Trisglycine 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

4
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
manufacturers 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 precoated 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 3,30 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 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 Students ttest 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/). The purpose of this analysis
was to determine if levels of haemoglobin (the dependent variable) in burns

5
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 variables) 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

6
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 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
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 nonsystemic 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 abundance between samples prior (Fig. 3A; 976.0 RBCs/mL
762.1; range 04000) and
subsequent (Fig. 3B; 10.0 RBCs/mL
7.07; range 030) 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

7
( p < 0.001). Based on this model, the haemoglobin levels 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 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. 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

8
increased fragility of circulating erythrocytes, both free haemoglobin and
contained within erythrocytes are increased substantially.

that

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.
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 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 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 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.

9
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 [2832]; however, other authors suggest that the
prolonged exposure to proteolytic and immunosuppressive factors within the
fluid [3337] 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 reepithelialisa- 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. Until such time as

10
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 Childrens
Health Foundation.

Extra-large negative pressure wound therapy dressings for burns

Initial experience with technique, fluid management, and outcomes

Abstract

Objective: The use of negative-pressure-wound-therapy (NPWT)

is associated

with im- proved outcomes in smaller burns. We report our experience using extralarge (XL) NPWT dressings to treat

15% total body surface area (TBSA) burned and

describe our technique and early outcomes. We

also provide NPWT

exudate

volume for predictive fluid resuscita- tion in these critically ill patients.
Methods: We retrospectively reviewed patients treated with XL-NPWT from 2012 to
2014. Following excision/grafting, graft and donor sites were sealed with a layered
NPWT dress- ing. We documented wound size, dressing size, NPWT outputs, graft
take, wound infections, and length of stay (LOS). Mean NPWT exudate volume per
%TBSA per day was calculated. Results: Twelve burn patients (mean TBSA burned
30%, range 1560%) were treated with XL- NPWT (dressing TBSA burned and skin
graft donor sites range 1744%). Average graft take was 97%. No wound infections
occurred. Two
compared to

patients had burns

50% TBSA and their LOS was reduced

ABA averages. XL-NPWT

outputs peaked at day 1 after grafting

followed by a steady decline until dressings were removed. Average XL-NPWT

dressing output during the first 5 days was 101

66 mL/%BSA covered per day. 2

patients developed acute kidney injury.

Conclusion:

The use of

XL-NPWT

to

treat extensive burns is

feasible with

attention to application technique. NPWT dressings appear to improve graft take,

and to decrease risk of infection, LOS, and pain and anxiety associated with wound
care. Measured fluid losses can improve patient care in future applications of
NPWT to large burn wounds.

11

1. Introduction
Negative pressure wound therapy (NPWT) has expanded our ability to treat acute
and chronic wounds and belongs in the standard repertoire of every surgical
department [1]. NPWT provides a sterile occlusive wound healing environment
that promotes re-epithelialization [2]. Studies have shown that wound healing
improves due to increased diffusion of blood and nutrients to the healing site [3,4].
For patients with large burn wounds, the inability of deep wounds to epithelialize is
a significant source of infectious morbidity and mortality [5]. Dermal vessels are
damaged leading to a depletion of erythrocytes and immune cells in the wound
bed [69]. In addition, burns exceeding 15% TBSA result in the release of
inflammatory mediators that provoke immunosuppression, increased vascular
permeability and fluid shifts to extravas- cular spaces [1012]. As a sterile
occlusive dressing that promotes re-epithelialization, NPWT has the potential
to reduce infectious complications and improve wound healing in burn patients.
The widespread use of NPWT in burn surgery has been limited, however, likely
due to challenges with dressing application in this patient population.
A sterile wound environment, proper wound healing and adequate fluid
resuscitation are mandatory for survival in critically ill burn patients. Although
the latter is more crucial in the initial phase of large burns, fluid replacement
regains importance after surgical debridement and autologous skin grafting due to
enlargement of the affected body surface area from the donor site [13]. In current
clinical practice, insensible fluid losses are estimated using body weight, heart rate,
blood pressure, urine output, laboratory values, and other param- eters. These
attempts to estimate the patients fluid status and volume repletion are often
imprecise and can lead to pulmonary edema or renal failure with life-threatening
consequences [1417]. In order to limit the effects of dramatic shifts in wound
area, a staged approach to excision and autografting is often implemented,
which on the other hand has the potential to prolong hospitalization and increase
the risk of wound infections and other nosocomial complications [18]. By
quantifying wound exudate, NPWT has the potential to improve one of the most
vexing problems in current burn care: fluid management. More informed fluid
management may lead to reduced pulmonary and renal complications.
Kamolz et al. recently reported about feasibility of NPWT for large-scale burns
exceeding 25% TBSA burned in 37 patients [19]. Although this excellent study
clearly demon- strates beneficial effects of NPWT
for severely burned
patients and thus increases the demand for more evidence supporting this
technique, detailed data regarding complica- tions and fluid losses associated
with NPWT were not provided.
Several studies have demonstrated that NPWT is capable of reducing infection
rates and enhancing graft take after burn wound excision and autografting [1,20].
These studies, however, are limited to small burns that do not exceed an

12
average of 10% TBSA. Based on these studies it is impossible to know if NPWT has
a beneficial effect on the systemic alterations associated with larger burns.
Finally, there is some data to suggest that NPWT has a beneficial effect on reepithelialization of skin graft donor sites [21]. Nevertheless, this data is based on
an animal study and case reports involving small wounds.
The aim of this study was to evaluate our outcomes using extra-large NPWT (XLNPWT) dressings in burn patients with wounds 15% TBSA burned, and provide
a description of technical refinements that make dressing application feasible for
large burn wounds. A secondary aim was to quantify wound exudates using
NPWT in an effort to predict estimated fluid losses after excision and
autografting. We sought to compare these outputs to estimated insensible losses
using standard calculations, and to suggest measuring NPWT exudate as a
useful tool for clinicians managing burn patients.

2.

Patients and methods

An IRB-approved retrospective review was performed of patients admitted to

our burn center from April 2012 to April 2014. We included patients that received
NPWT after burn excision and grafting with wounds
15% TBSA burned.
Wounds were covered with xenografts or autografts using a power dermatome
(0.100.12 inches) and 2:1 or 3:1 mesher. Patient records were reviewed to
determine graft take (%), donor site healing (time to 95% re-epithelialization),
ventila- tion days, complications, length of stay (LOS), and volume of wound
exudate. Dressing size was measured manually with a conventional ruler and also
calculated as a percentage of the TBSA via Lund and Browder method [22].
Estimates of insensible losses were calculated by the formula of DuBois and
DuBois involving body weight and height [23]. Wound exudate was gathered
daily and provided in total (grafted + - donor sites) as well as separately for graft
and donor sites. Wound exudate output was calculated in mL per percentage of VAC
covered body surface area per day and given in average of the first 5 days after
grafting as well as for each of the first 5 days separately. NPWT dressings placed
over xenografts were excluded from these calculations to simplify our analysis.
Daily burn related evaporative losses (BREL) were calculated according to the
following formula [24]:
BREL = (25 + %TBSA)

(BSA in cm2)

24.

Hospital LOS was compared with average LOS provided by the American Burn
Association (ABA) according to patients age and TBSA burned.

3.

Negative pressure wound therapy dressing application

NPWT dressings were applied to both burn wounds and donor sites. In
the
majority of
cases, tangential excision and autografting was performed. Less

13
often, fascial excision with or without grafting was performed. Xenografts were
used in certain cases to test the wound bed or provide temporary biologic
coverage. Graft recipient sites were covered with non- adherent fine mesh gauze (NTERFACE, Winfield Laboratories, Richardson, Texas, USA and/or Xeroform Covidien,
Mansfield, MA, USA) and donor sites with thin silver-impregnated non- adherent
foam (Mepilex Ag, Mo lnlycke Health Care, Gothen- burg, Sweden). All surfaces
were then covered with NPWT dressings consisting of polyurethane foam
(V.A.C. Granu- Foam, KCI, San Antonio, TX), transparent self-adhesive drape (V.A.C.
Drape, KCI, San Antonio, TX and/or Ioban, 3 M, St. Paul, MN) and a suction pad
(Sensa T.R.A.C. Pad, KCI, San Antonio, TX).
Recognizing that prolonged maintenance of seal was critical to the success of
the NPWT, multiple application strategies were employed. On extremities, a
seal was obtained by sandwiching the limb between large sheets of occlusive
dressing (Fig. 1). Tincture of benzoin was used to prepare the dried skin prior to
application of adhesive drapes. Wall suction was utilized to provide the initial
seal and the most difficult areas were sealed last, after wall suction was
applied, so an immediate seal was obtained. In areas of shear or moisture (e.g.
perineum, non-excised burn, open tissue), a double layer of occlusive dressing was
applied and secured by stapling or suturing to the moist tissue (Figs. 25).
Suction pads were applied to the most dependent areas. Negative pressure was
provided by 26 suction pumps (V.A.C. Therapy Unit, KCI, San Antonio, TX) per
patient. In general, 1 pump was used per 9% TBSA body area as defined by Lund
and Browder [22]. For example, one pump was used per arm and two pumps per
leg. NPWT was applied at
125 mmHg. Range of motion was permitted at postopera- tive day 13 with NPWT
turned down to
50 mmHg, at the discretion of the attending burn surgeon.
When a seal was unable to be maintained via the KCI device, despite standard
trouble shooting, that suction pad was switched to wall suction. Dressings
were taken down in the operating room 57 days after application.
A fluid replacement guideline was developed and followed after the first case.
When XL-NPWT output was predicted to be >3 L/d (based on current output),
output was replaced with lactated ringers solution in a 1:1 ratio calculated every
4 h shift. Electrolytes were checked twice daily, and corrected per standard ICU
management.

4.

Results

Twelve patients were included in this study with an average age of 35.5 years
(median: 28, range: 1863). The mean burn size was 29.6% TBSA burned (median:
25, range: 1560%). Six patients (50%)
also suffered
inhalation
injury.
Mechanical ventilation was undertaken in 11/12 (92%) patients for an average
of 16.5 days (median: 15, range 240 days). Initial wound debridement was

14
performed on average 4.4 days after burn (mean: 5, range 27 days). Xenograft
application was performed in 6 cases as a temporary measure. Mean time from
burn to autografting was 7.75 days (median: 7.5, range: 217 days). TBSA covered
with a NPWT dressing was on average
35.1% TBSA (burned and skin graft donor sites, median: 32, range: 1744%).
Average graft take was 97% (median: 100, range: 85100%). Donor sites were reepithelialized after an average of 11.25 days (median: 11, range: 1014 days),
although this information was only available for 4 patients.
Two patients (17%) developed acute kidney injury (AKI), of which
one
occurred subsequent to NPWT. Hematoma occurred in 1 patient under a
donor site dressing within the first 12 h after surgery, undergoing evacuation
under the dressing. The dressing was then re-sealed with no subse- quent
complication. LOS averaged 37.9 days (median: 32.5, range: 1966). Two patients
with burns exceeding 35% TBSA had shorter LOS than the average LOS according
to ABA Burn Repository national averages (39 vs. 62 days and 50 vs. 68 days,
respectively). An overview of all patients is given in Table 1. There were no
wound infections and every patient survived.
Average NPWT dressing output during the first 5 days after grafting was 101
66 mL/% BSA covered per day. Donor site output was more than double
recipient site output. Average output of recipient sites was 61 37 mL/%BSA/day
and average output of donor sites was 132 83 mL/%BSA/ day. Peak output was
observed on the first day for both combined and recipient sites (154 85 mL/
%BSA/day and
93
30 mL/%BSA/day) and over the first two days for donor sites (175
%BSA/day on day 1 and 169 92 mL/

92 mL/

%BSA/day on day 2 after grafting) (Table 2 and Fig. 6). Of note, the reduced
output observed on the day of NPWT application (day 0) is mainly attributed to
the time point of surgery, as outputs were usually not recorded until late
afternoon. Mean calculated BREL was 2519 mL/day. BREL did not correlate with
NPWT dressing output during the first 5 postoperative days, and was typically
higher than peak NPWT output.

5.

Discussion

This study demonstrates that NPWT is safe and effective in burn patients with
wounds exceeding 15% TBSA burned. Although there is a steep learning curve,
with attention to proper technique a seal is possible in larger burns previously
thought to be unamenable to NPWT. After excision and autografting, NPWT
seemed to prevent wound infection and promote excellent graft take and reepithelialization of donor sites. In addition, fluid management decisions were
informed by NPWT output measurements that revealed a mean output of 101 mL/

15
%BSA/day and a peak of 154 mL/%BSA/day on day 1 after grafting. These
measurements may be useful to estimate fluid losses in cc per %TBSA (both burned
and skin graft donor sites) during post surgical XL-NPWT in burn patients, and
perhaps following excision/autografting in general (without the use of NPWT).
In 1998, Schneider et al. introduced NPWT to secure skin grafts to difficult
recipient beds [25]. Since then several authors have reported their experience
with NPWT for skin graft fixation and revealed improved graft-take and accelerated healing [26,27]. With respect to burn wounds Petkar et al. applied NPWT to
30 patients with a mean wound size of 244 cm2 and revealed an average grafttake of 96.7% [28]. Waltzmann et al. reported a mean graft take of 99.5%, in 76
patients with an average burned surface area of 367 cm2 [29].
In our study the average graft take was 97%, thus comparable to results
achieved in the literature. Wound size, however, was significantly higher, as none
of the aforemen- tioned studies involved wounds that exceeded 10% TBSA
burned. This demonstrates that the efficacy of NPWT is not limited to small burns.
Moreover, NPWT may be more effective in larger burns where a higher rate of graft
loss has been the norm due to shear forces (especially in mobile joints, the
posterior trunk, the perineum, etc.) (Fig. 7).
Attempts to accelerate re-epithelialization of autograft donor sites include
various types of dressings and applica- tions. With respect to NPWT, Genecov et
al. assessed healing of donor sites histologically at day 7 after grafting in 4 pigs
and 10 humans and demonstrated a statistically significant increase in the rate
of re-epithelialization due to
NPWT application [21]. In our study complete
healing of donor sites was obtained an average of 11.25 days after skin graft
harvest. Wound infections were not observed and apart from NPWT removal a
maximum of two dressing changes were necessary.
It is important to note that the wounds treated in this study (including both burns
and donor sites) were significantly larger than those included in
the
aforementioned studies. The only studies that address wounds in comparable
size were performed by Kamolz et al., Chong et al. and Low et al. [19,30,31].
By applying NPWT to 37 patients with at least 25% TBSA burned, Kamolz et al.
were able to demonstrate favorable results regarding graft-take, infections and
survival as well as its feasibility of skin graft fixation in areas of irregular
wound surfaces or subject to movement. Although these results are well in
accordance to our findings and clearly substantiate beneficial attributes related to
NPWT for large- scale burns, fluid losses were not evaluated in this study. In
contrast Chong et al. and Low et al. (both from the same unit) demonstrated the
applicability of total body wrap NPWT in burns of 2260% TBSA and provided
information about the body fluids collected [30,31]. After wound excision
and autografting, limbs and trunks of 8 patients (combining both studies) were
sandwiched with large polyurethane dressings.

16
A thin strip of sponge was placed in the dependent part of the dressing only, and
therefore this application is quite different from traditional technique and may
not provide the same fixation of grafts. Average daily exudate was collected in
one study [30] for 3 patients and ranged from 526 to 1211 mL during the first 3
postoperative days, but output did not seem to correlate with TBSA (2260%).
The authors concluded that total body wrap NPWT is feasible and improved
healing, patient comfort and management of the extensively burned patient.
Our study substantiates these findings. Although applied in conventional
technique with sponges that covered the entire wound area, sealing was
achieved by sandwiching extremities and by stapling or suturing occlusive
drapes in areas of shear or moisture. To provide initial seal wall suction was
utilized and the most difficult areas were sealed at last. Furthermore, fluid
resuscitation was beneficially assisted by VAC output measurements. In detail,
average fluid loss during the first 5 days after grafting was 100.98 ml/%BSA/
day, with a peak on day 1 and subsequently declined over the following days after
grafting. In this context, it is important to mention, that the reduced output on
the day of VAC application (day 0) is mainly attributed to the time point of
surgery and should be considered for half or even less of the day of application.
Lamke et al. evaluated evaporative water losses depending on burn depth in an
experimental study and found superficial second degree burns to be equal to
donor sites and both losing higher amounts of fluid than third degree burns [32].
In the same study wound evaporation was reduced by 8085% after autologous
skin grafting making water loss of donor sites after grafting about 3 times higher
compared to grafted sites (1.2 vs 0.36 mL/cm2/day).
Our study showed comparable results, as donor sites yielded more than
twice as much NPWT output compared to grafted sites. Interestingly, by
calculating our NPWT output in mL per cm2 of affected BSA per day (data not
shown), grafted sites have similar but donor sites much less output compared to
the studies of Lamke et al. (0.36 vs. 0.35 mL/cm2/day and 0.7 vs.
1.2
mL/cm2/day, respectively). The latter might be
attributed to the inner layer of the donor site dressing we used that may contain
fluid exudate despite suction. This effect might be irrelevant on grafted sites as a
seal is predominantly provided by skin grafts and the additional contribution of
the sponges is insignificant.
Proper fluid resuscitation
is
necessary to
prevent life- threatening
complications, like acute kidney injury (AKI) and lung edema. AKI is seen more
frequently during the resuscitation period and incidence varies between 14.6 and
39.1% among burn patients [33,34]. In contrast lung edema occurs more often
after than during the fluid-resuscitation phase of burn [35]. Clinical studies
suggest, that lung edema is more related to inhalation injury, followed by

17
hypoproteinemia then by fluid overdose or retention, respectively [3638].
Nevertheless, fluid restriction (a dry or conservative strategy) has been proven
effective to overcome lung injury and acute respiratory distress syn- drome
(ARDS) [39].
With respect to complications associated with adequacy of fluid resuscitation, 2
patients in our study developed AKI. It is possible that lack of experience with
large-scale NPWT and consequent underestimation of fluid resuscitation may
have attributed to the occurrence of AKI. On the other hand none of our patients
developed clinically significant lung edema and mechanical ventilation could be
discontinued earlier com- pared to data from burn patients provided in the
literature [40].
There are several limitations to our study. To prove an improvement in
outcomes due to NPWT, a control group would be necessary. Our early results
are so encouraging, however, that we are reluctant to recommend a randomized
study. The only comparisons that were feasible involved LOS and BREL with ABA
registry data and the formula according to Warden et al., respectively [28]. All
other advantages of XL- NPWT are only suggested in accordance and compared
with data provided in the literature. In addition, our determination of reepithelialization was based on subjective assessment in a non-blinded fashion.
Therefore, further studies are needed to substantiate our findings and verify the
beneficial attributes of XL-NPWT in burn patients.
The aim of this study was to demonstrate the feasibility of XL-NPWT in burn
patients, describe our technique, demon- strate our early results, and assess
wound exudate outputs. By providing the average fluid output related to wound
area covered with NPWT, further studies involving XL-NPWT can be performed
with more proactive fluid replacement strate- gies.

6.

Conclusion

XL-NPWT is safe and effective in large burns. In addition to promoting

epithelialization of both grafted and donor sites, we observed excellent graft
take, no wound infections and significantly less anxiety and pain. Following
excision and autografting, fluid management was informed by NPWT output
measurements, potentially reducing the risk of AKI and pulmonary edema. As a
result of the above findings, XL- NPWT has now been incorporated into the
standard of care for large burns at our institution.

18
Anterolateral thigh myocutaneous aps as the preferred aps for
reconstruction of oral and maxillofacial defects

abstract

Background: The anterolateral thigh myocutaneous ap is one of the most

commonly used aps in reconstructive procedures, but its application in oral
and maxillofacial defects has not been fully determined. Herein, we summarize
the application of 1212 anterolateral thigh myocutaneous aps in the repair of oral
and maxillofacial defects and examine their benets in maxillofacial reconstruction
of these defects.
Methods: Patients were recruited from February 2002 to June 2013 in the
Department of Oral and Maxillofacial Surgery of Central South University. All
patients
underwent
reconstructive
surgery employing anterolateral thigh
myocutaneous aps. Patient ages ranged from 6 to 82 years with a mean age of
51.2 years. There are 1015 aps showing single lobe and 197 aps showing a
multi-island pedicle and one of which carries the iliac bone. The largest area among
the single aps was 28x12 cm2, and the smallest was 3x2 cm2.
Results: Among the 1212 transferred aps, 1176 survived and 36 showed necrosis,
a survival rate of about 97.0%. The common complications at ap donor site
were poor wound healing (10.1%), localized paraesthesia (50.1%), and altered
quadriceps force (11.0%). No cases presented with local serious com- plications,
and 90% of patients achieved good functional recovery and aesthetically
acceptable results after reconstruction of oral and maxillofacial defects at
various locations using anterolateral thigh myocutaneous aps. The time (23-121
min; average 51 min) for anastomosis of one vein and one artery was signicantly
less than that for two veins and one artery (45-153 min, average 83 min; p
0.0003), which indicates one vein anastomosis can signicantly reduce the
operating time.
Conclusion: The anterolateral thigh myocutaneous aps can be easily obtained and
can provide a good amount of muscle for lling dead space and fascia lata. These
aps can be prepared into a separate fat ap, multi-island fascia with iliac bone,
and other composite pedicle aps to meet the various re- quirements of oral
and maxillofacial defects. The subcutaneous fat thickness of the anterolateral area
can vary considerably and thus can be used to repair defects requiring different
ap thickness. Therefore, the anterolateral thigh myocutaneous free aps are
more suitable for oral and maxillofacial defects than other aps.

1.

Introduction

The oral and maxillofacial areas are extremely important re- gions for both
aesthetic and functional reasons. If defects at these regions are not promptly

19
repaired, the adverse effects can include not only bad speech and chewing and
swallowing disorders but also psychological problems caused by disgurement.
With the development of free aps and microsurgical tech- niques, a variety of free
aps have become the most commonly used tissue source for repair of maxillofacial
defects (Crow and Crow, 1976; Bianchi et al., 2008). More than a dozen free aps
can be considered, and knowing how to choose the right ap for reconstruction is an
important key step for successful repair (Futran and Mendez, 2006). The ideal
free aps for oral and maxillofacial defects should have the following features:
simple preparation, constant vascular anatomy, a sufcient amount of diverse
tissues, few donor site complications, and vascular diameter similar to that of
neck and maxillofacial vessels. In- vestigators around the world have made great
efforts to nd the best free aps for the repair of maxillofacial defects (Gedebou et
al., 2002).
The application of an anterolateral thigh ap was rst reported by Song et al. (Song
et al., 1984), anterolateral thigh aps offer many features and can be prepared as
perforating aps, fat aps, fascia aps, muscle aps, various chimeric aps, and
others (Koshima, 2000; Demirkan et al., 2000; Koshima et al., 1993; Fang et al., 2013a,
2013b). Therefore, these aps have become one of the most commonly used aps for
the repair of defects in the head and neck, limbs, torso, and other body parts.
However, the clinical value of anterolateral thigh myocutaneous aps in oral and
maxillofacial surgery has not been widely recognized, and a large cohort-based
report of the application of anterolateral thigh myocutaneous ap in oral and
maxillofacial defects has not been reported. Here, we collected and summarized data
of 1185 patients who underwent reconstruction of oral and maxillofacial defects
using anterolateral thigh myocutaneous aps from February 2002 to June 2013. Our
results clearly demonstrate that anterolateral thigh myocutaneous aps are the
preferred aps for oral and maxillofacial defects.

2.

Materials and methods

2.1. Patients

Patients were treated from February 2002 to June 2013 in the Department of Oral
and Maxillofacial Surgery of Second Xiangya Hospital. All 1185 patients (1212 aps)
underwent reconstruction of defects caused by tumour resection or trauma using
anterolateral thigh myocutaneous aps. 904 cases were male and 281 cases were
female. The youngest was 6 years old, and the oldest was 82 years. The mean age was
51.2 years. Among all of the transferred aps, 1015 were single lobe, and 197 were
multi-island pedicle aps, of which one carried the iliac bone. The largest area among
the single aps was 28x12 cm2, and the smallest was 3x2 cm2. Postoperative followup was performed for 1092 patients. Follow-up time varied from 5 to 84 months, and
the mean follow-up time was 32.6 months. Basic information for all patients including
defect side, ap size and type, recipient vessel processing method, donor complications,
and postoperative quality of life were recorded and statistically analysed.

20

2.2. Surgical technique

The incision line is parallel to the iliac patellar connection line at 2 cm to the inside of
the iliac patellar connection line (Fig. 1). The position and distance of the incision line
are adjusted according to the width of the prepared ap. If the ap is wide, the incision
line is moved to the inside; if the ap is narrow, the incision line is moved outward.
Retrograde anatomy is performed to obtain appropriate vessel and vascular pedicle as
well as its concomitant vastus lat- eralis and motor nerves, and all the tissues are
appropriately restored. Different amounts of vascular pedicle muscle tissue are used
to ll the dead space. When repairing complicated defects, we can also take advantage
of the lateral femoral circumex artery, which is characterized by multiple perforating
branches, to prepare separate fat aps, fascia, muscle aps, or iliac pedicles of a multiisland composite ap (Case 1).
The donor wound is closed by direct suture after preparation of complete aps. When
the cut ap is too wide or under too much tension, the ap can be prepared in a
region above or below the anterolateral area by cutting full-thickness skin without
expanding the donor site. We also can use the lateral femoral circumex artery to
prepare the anterolateral thigh ap pedicle or tensor fascia lata ap and close the
wound by local pedicle ap repair.
2.3. Statistical analysis
Data were analysed using SPSS 16.0 software (SPSS, Inc., Chi- cago, IL, USA). c2 test
or Fishers exact test were used to analyse the data. A p value <0.05 was considered
to indicate a statistically signicant difference.

3. Results
Among all 1212 anterolateral thigh myocutaneous aps trans- ferred, 1176
survived and 55 showed postoperative vascular crisis. Among those 55, 19 survived
completely after treatment, whereas 36 showed necrosis. The overall survival rate was
97.0% (1176/1212; Table 1).
We compared the ratio of vascular crisis between the cases of two vein
anastomosis and cases of one vein anastomosis. Two vein anastomosis was performed
with 1091 aps, and 39 cases showed venous crisis, 26 of which showed necrosis.
Eleven cases showed crisis within 12 h after surgery, and 28 cases showed crisis
beyond 12 h after surgery. The incidence of venous crisis was 3.6% (39/1091), and
the survival rate after treatment was 33.3% (13/39). Necrosis occurred in 2 of 121
cases (1.7%). The survival rate after treatment was slightly higher in the one vein
anastomosis group compared to that in the two vein anastomosis group. However,
the incidence of venous crisis, the survival rate after treatment, and the rate of
venous crisis resulting in ap necrosis were comparable between the groups (p >
0.05; Table 2). The average micromanip- ulator processing time for two vein
anastomosis was 83 min (range, 45-153 min). The average micromanipulator
processing time for one vein anastomosis was 51 min (23e121 min), which was
signicantly shorter than that for two vein
anastomosis (p 0.0003). No
case showed limited knee movement, compartment syn- drome, or other serious

21
complications (Table 3). Postoperative follow-up was performed in 1092 patients
(1092/1185 92.2%). Follow-up time ranged from 5 to 84 months, and mean
follow-up time was 32.6 months. Questionnaires, oral eval- uation, or other methods
were used to assess patients quality of life and obtain information about their
speech, swallowing, chewing, and appearance. In addition, 150 patients with
tongue, 50 cases with buccal carcinoma, 20 cases with oropharyngeal cancer, and 20
cases with mouth cancer were randomly selected and further analysed 1 year
after surgery. Patients were grouped according to chewing function as normal, only
soft food, and non-masticatory function by considering foods that could be chewed
by patients. Patients were also grouped according to normal verbal communi- cation,
barely able
to verbally communicate and unable to communicate verbally
according to the clarity of pronunciation and their ability to be understood. Patients
were also grouped according to the patients opinions of their change in appearance as
good and beautiful, acceptable, and unacceptable. Because the degree of mouth
opening in patients with buccal cancer is an important in- dicator of the quality of
life, we measured the buccal opening de- gree and divided cases into 3 cm, 1e3 cm,
and <1 cm. The results showed that chewing, swallowing, speech, appearance, and
other oral maxillofacial functions were perfectly recovered in 90% of patients with
tongue, cheek, oor of the mouth, and other oral and oropharyngeal cancers through
anterolateral thigh myocutaneous ap reconstruction (Table 4).
Discussion
Although the advantages and application of anterolateral thigh aps have been
widely reported, many investigators believe that the thickness of fat in the
anterolateral thigh ap is unsuitable for repair of oral and maxillofacial defects. Our
results obtained for a large cohort clearly demonstrate that thicker aps are suitable
in the reconstruction of defects in oropharynx, cheek, and other areas and for the
recovery of related functions. We also found that fat thickness is signicantly
different in the upper and lower regions of the anterolateral area, and thus we can
prepare the aps according to varying requirements of the defects to be repaired. In
addition, thick aps can be processed into thin aps (Case 2). Some in- vestigators
considered that the preparation of an anterolateral thigh ap may fail due to
variation of perforating branches (Wei et al., 2002). Our data show that none of the
1212 anterolateral thigh myocutaneous aps failed due to variation of perforating
branches. When preparing the anterolateral thigh myocutaneous aps, we retained a
certain amount of muscle sleeves around the perforating branches to ensure that the
perforating branches were not damaged, because even smaller perforating branches
can pro- vide the blood supply to the ap. In addition, the perforating branches
surrounding muscle tissue can ll the dead space and serve to prevent effusion and
infection.
Wong et al. (Wong and Wei, 2010) and Kimata et al. (Kimata et al., 1997) reported
that the anterolateral thigh ap has advan- tages in head and neck applications. In
2000, Demirkan et al. (Demirkan et al., 2000) were the rst to report that
anterolateral thigh myocutaneous aps not only
have reduced potential for
damage to branches but also that surrounding muscle can be used to ll dead space.
Subsequent studies also show that anterolateral thigh myocutaneous aps have
advantages in the preparation process and that it is necessary to include muscle
tissue when repairing large defects (Kuo et al., 2002; Wong et al., 2009a, 2009b; Liu et

22
al., 2010). However, the numbers of cases included in previ- ous reports are relatively
small, and the application of anterolateral aps in oral and maxillofacial defects was
not examined. The present study summarizes the outcomes of the application of 1212
anterolateral aps in oral and maxillofacial defects and their preparation process
and provides useful information regarding the application of anterolateral aps in oral
and maxillofacial defects.
The organizational structure in the oral and maxillofacial area is very complicated, and
the thickness and volume of tissue required for reconstruction varies greatly. In
addition, the presence of the mandible, zygomatic, zygomatic arch, and other bone
tissues in these area are conducive to the formation of void spaces in the skull base,
zygomatic, lateral pharyngeal, and submandibular areas. The thickness of the
subcutaneous fat in the upper and lower portions of the anterolateral area differs
considerably, and this area contains skin, muscle, and other tissues, which can satisfy
the requirements for repairing oral and maxillofacial defects (Gong and Wu, 2013).
Although the free forearm ap is the preferred ap for buccal and oropharyngeal
defects, the insufcient amount of forearm ap tissue increases the likelihood of
collapse at the repaired site. Thus, the use of a forearm ap is not an ideal method in
terms of both appearance and functional considerations (Fang et al., 2013a, 2013b;
Seikaly et al., 2003). In the present study, we prepared aps from the up- per
anterolateral area regularly for oropharyngeal or cheek defects.
Immediately after surgery, the transferred aps appeared slightly bloated, but
vocal activity, swallowing movement, and tongue pressure can re-shape the form of
the aps. The recon- struction of the cheek or the oropharynx was able to achieve
good shape and restoration of function within 3e6 months after surgery (Cases 3 and
4). Repairing the tongue or oor of the mouth after cancer treatment often requires a
thin subcutaneous fat ap, which can be obtained under the anterolateral area (Case
5).
Careful control of the length of a vascular pedicle is needed, because a vascular
pedicle that is too long or too short will cause postoperative venous crisis (Yu et al.,
2009). According to our re- sults from using 1212 aps in the repair of oral and
maxillofacial defects, when there is no obvious damage in the ipsilateral neck vein,
a vascular pedicle length of 8 cm is appropriate. When reconstructing a defect in
the oor of the mouth, the vascular pedicle length can be shortened as needed. For
the repair of a cheek defect, a vascular pedicle length of 10 cm is appropriate. When
reconstructing a defect in the oropharynx or upper jaw, a vascular pedicle length of 12
cm is appropriate. We used upper and lower extremity supercial veins (saphenous
vein, cephalic vein, small saphenous vein or external jugular vein anastomosis) for
bridging via arterial and (or) venous anastomosis, if the vascular pedicle ap length
was insufcient and achieved good results. Bridging anas- tomosis was performed in
25 cases in this study, and all aps sur- vived without vascular crisis.
Gedebou et al. (Gedebou et al., 2002) reported the application of 1284 free anterolateral
aps at various body sites and showed that the main drawback of the anterolateral
thigh ap is the inability to carry bone tissue. In our study, one double island ap
carried one iliac; the ap area was 7
12 cm and the bone portion was
approximately 6 2 2 cm in size. The ap was used to repair a defect in an advanced
mandibular buccal cancer patient. Survival of the ap was good, and satisfactory
recovery was achieved. However, the transferred bone tissue should be less than 10 cm

23
in length. We also used a separate fat-containing ap or fascia of a multi-island
pedicle anterolateral thigh myocutaneous ap for mandibular reconstruction in oral
cancer patients, and a ap of fat or with fascia covering the titanium surface effectively
prevented postoperative titanium plate exposure and increased the fullness of the
cheeks.
The anterolateral (musculocutaneous) pedicle ap usually con- tains one artery
and two veins. Anastomosis was performed in all arteries and veins at the beginning
of this study. By contrast, from February 2013 to June 2013, anastomosis was
performed in one artery and one vein in a total of 121 cases (Ren et al., 2013). We
believe that one vein anastomosis has many advantages, and the two most
prominent advantages are: (1) this creates an early warning before crisis and
improves the skin ap survival rate after crisis (early warning means venous
crisis can be discovered earlier), (2) signicantly shorter ap anastomosis time.
In this study, the most common donor site complications included poor
wound healing, localized paraesthesias, and decreased quadricep strength, among
others. The main causes of the above complications are known to be excessive
suture tension and intraoperative injury of the anterolateral thigh muscle motor
nerve and cutaneous nerve (Lin et al., 2006; Choi et al., 2007; Wong et al., 2009a,
2009b). Therefore, during ap preparation, we should protect the anterolateral
cutaneous nerve and vastus lateralis mo- tor nerve as much as possible and avoid
large suture tension to further reduce donor site complications.

5. Conclusion
Anterolateral thigh myocutaneous aps can be easily obtained and can provide a good
amount of muscle for lling dead space and fascia lata. These aps can be prepared
into a separate fat ap, multi-island fascia with iliac bone, and other composite
pedicle aps to meet the various requirements of oral and maxillofacial defects. The
subcutaneous fat thickness of the anterolateral area can vary considerably and thus
can be used to repair defects requiring different ap thickness. Therefore, the
anterolateral thigh myocutaneous free aps are more suitable for oral and maxillofacial
defects than other aps. In addition, compared to two vein anas- tomosis, one vein
anastomosis is associated with a similar ap survival rate but signicantly shorter
vascular anastomosis time.

Reconstruction of complex soft-tissue defects in the extremities with chimeric

anterolateral thigh perforator ap

abstract
Introduction: The reconstruction of extensive three-dimensional defects in the
extremities is a difcult challenge. Many attempts have been made to reconstruct
such defects using the chimeric ap concept, enabling aps with larger surface

24
areas to be used while maintaining economical tissue use. The ante- rolateral
thigh (ALT) chimeric ap is one of the most useful tools for the reconstruction of
complex three- dimensional defects in the extremities.
Methods: From January 2010 to March 2012, Twenty-two patients underwent
extremity reconstruction using chimeric ALT perforator aps, which consists of a
skin component on its isolated perforator and a portion of the fascia and muscle
aps on the same pedicle from the descending branch of the lateral circumex
femoral artery (LCFA). The defects were in either a lower (n 10) or an upper
extremity (n 12). The area of the soft tissue defects ranged from 43x35 cm to
19x9 cm (mean, 25x18 cm), containing extensive, irregular, ring-like soft tissue
defects or degloving injuries.
Results: The mean dimension of skin ap was 19.8x 11.2 cm. The mean dimension
of fascia ap was 8.9
7.1 cm. The mean dimension of muscle ap was 11.1x 7.5
cm. No total ap loss occurred. One patient presented with venous thrombosis,
and re-anastomosis and vein grafting were performed. Two cases exhibiting
partial skin graft loss at the site at which the fascia ap was inset were treated
via secondary skin grafts. During a follow-up period of 18 monthse30 months,
patients were satised with the functional and aesthetic outcome. No serious
donor-site complications occurred.
Discussion: Chimeric anterolateral thigh perforator ap can be one of the best
choice for reconstruction of complex soft-tissue defects in the extremities.
Conclusions: The various tissue components and maximal freedom offered by
chimeric tissue aps associated with the same descending branch of the LCFA
provide versatile coverage of large, complex, and irregular soft-tissue defects in
the extremities.

1. Introduction
The primary goal of extremity reconstruction is to restore and maintain extremity
function, and skeletal reconstruction resulting in stable soft-tissue coverage is
essential for achieving this goal. In cases of initial reconstruction failure, additional
tissue and func- tionality might be lost [1]. Therefore, it is crucial to devise an
appropriate treatment plan. However, reconstructing complex in- juries of
extremities containing extensive, irregular, ring-like soft tissue defects or degloving
injuries remains a challenge for recon- structive microsurgeons, as very few aps
contain large amounts of tissue and multiple components are required to fulll the
func- tional and cosmetic requirements for correcting the recipient defect while
inducing minimal donor-site morbidity.
Since its rst description by Song et al., in 1984, the anterolateral thigh (ALT) ap has
become one of the most popular aps for extensive soft-tissue defect reconstruction
[2] because of its large skin area and reliability [3]. In addition, the ALT ap is
versatile in that multiple tissue components can be harvested in various
combinations and congurations, thus fullling all of the re- quirements for

25
reconstruction [4].
For
extensive, complex, and irregular wounds in
the
extremities, the chimeric ALT perforator ap is among the most popular members
of the perforator ap family. A large tissue ap can be designed that includes
multiple tissue components, such as muscle, fascia, and skin, each of which is
supplied by a separate perforator based on the same lateral circumex femoral
artery (LCFA) [5]. This chimeric ap enables the use of multiple, spatially independent
components of various tissue types to reconstruct complex three-dimensional
defects in the extremities via a single vascular anastomosis. From this chimeric ap,
a small area of skin at the donor site can be harvested to yield the largest amount of
tissue possible for the reconstruction of massive defects in the extremities with
minimal loss of function at the donor site.
In this report, we describe the use of this technique for har- vesting a chimeric ALT
ap consisting of a skin component isolated on its perforator in combination with a
portion of the fascia and/or muscle aps, all of which are pedicled on the same
descending branch of the LCFA, for
the reconstruction of complex threedimensional and massive defects in the extremities.
2. Materials and methods
From January 2010 to March 2012, a total of 22 patients with large and complex
three-dimensional defects in the extremities underwent reconstructive procedures
that included chimeric ALT ap transfer. One patient was female, and 21 patients
were male. The patients mean age was 36.5 years (range, 16e57 years). The mean
body mass index was 25.2 5.1 (range, 15.5 to 42.2). The mechanisms of injury were
trafc accidents (n 8) and industrial injuries (n 14). The defects were in either a
lower (n 10) or an upper extremity (n 12). In these 22 patients, the area of the
soft tissue defects ranged from 43
35 cm to 19
9 cm (mean, 25
18 cm). In
ten, six, ve, and one patient, these aps were used for the treatment of a traumatic
limb amputation, a circumferential or near-circumferential de-gloving injury, or an
open fracture with an associated skin defect or to cover a soft-tissue defect without a
fracture, respectively. The recipient site was the lower leg in ve patients, the ankle
and foot in ve patients, the hand and ngers in six patients, and the forearm in six
patients. Details related to these patients are provided in Table 1.

2.1. Operative procedures

All of the aps were harvested in a similar manner. A line con- necting the anterior
superior iliac spine and the lateral border of the patella on the donor thigh with the
patient in the supine po- sition is drawn. The position of perforators usually
around the midpoint of this line should be mapped with a Doppler probe. A
longitudinal skin incision is made on the medial side of the ap. Then dissection is
carried out over the deep fascia until the perfo- rators are encountered. All the
perforating branches of the descending or transverse LCFA in their intramuscular or
intraseptal courses are identied at rst. Elevation of the ap is continued by
dissecting the descending branch toward the proximal and distal sides from the
division of the perforator. Then, a minimum of two perforators from the descending
branch of the LCFA are required to be preserved within the area of skin, fascia, or

26
muscle to be included in the ap, and the perforators are freely dissected until the
main trunk of the descending branch of the LCFA is reached (Fig. 1A). The chimeric
ALT perforator aps are designed and har- vested based on ndings during the
operation and the recon- structive requirements of the recipient sites; Flap
dimension is slightly larger than defect and slightly longer pedicle. These
chimeric aps should be designed to be included independent skin, fascia, or muscle
aps associated with the same descending branch of the LCFA. The skin and muscle
aps always contain individual paddles supplied by two separate perforators.
However, the fascia ap could be harvested by performing one of the following
three types of dissection depended on the different origins of pedicle: Type I, in
which the fascia ap is associated with one independent perforator that could be
rotated up to 180 ; Type II, in which the vascular pedicle of the fascia ap is a
separate branch from the source vessel or is located on a side branch of the skin ap;
or Type III, in which the many tiny perforators could not be freely dissected with the
fascia ap, and a fascial pedicle with a width of 2e3 cm is required to ensure a
sufcient blood supply (Fig. 1BeD). The muscle aps are harvested from the partial
vastus lateralis muscle, leaving the remaining muscle innervated and functionally
intact. The entire ap can then be isolated on the two or more perforators and the
descending branch of LCFA. The skin aps are used to cover important tissues,
such as vessels, nerves, and bones. The fascia aps are used to cover tendinous
tissues, followed by skin grafting. If the ap covered the soft tissue defect
insufciently or if severe contamination was caused by a bone defect or an empty
cavity, an additional muscle ap is designed for additional bulk to eliminate any dead
space or to enhance resistance to infection. If the injured extremities are associated
with soft-tissue defect and major arterial damage, the pedicle vessels of the ap can
be interposed into the vascular defect in the extremity to revascularise ischaemic
ex- tremities. The donor site can be closed primarily if the width was less than 8 cm,
or with skin graft if the width was over 8 cm.
The postoperative monitoring techniques include evaluation of color,
capillary
rell, turgor, surface temperature, presence of bleeding, skin graft adherence, and
auditory assessment of blood ow. The aps should be monitored frequently,
especially in the initial postoperative period. Emergent exploration and repair must
be performed soon after obstruction occurs if it is to be successful.

3. Results
The skin ap dimensions ranged from 33
13 cm to 8
7 cm (mean, 19.8 7.6
11.2 3.2 cm). The fascia ap dimensions ranged from 15
10 cm to 6
4 cm
(mean, 8.9 3.3 7.1 2.0 cm). The muscle ap dimensions ranged from 22
12 cm
to 6
4 cm (mean, 11.1 5.3
7.5 2.5 cm). There were 14 cases in which a Type I
ap was used, 5 cases in which a Type II ap was used, and 3 cases in which a Type III
ap was used (Table 2). The mean surgery duration for ap harvesting was 5.4 1.3 h.
One case exhibited a vascular complication (venous throm- bosis). In this case, a
thrombus was evident at the site of anasto- mosis. Therefore, the thrombus was
removed immediately, and re- anastomosis and vein grafting were performed.
Reperfusion was achieved, and only the partial margin of the 3
4 cm skin ap
exhibited necrosis. On postoperative day 14, this patient was treated with a

27
split-thickness skin graft. In total, two cases exhib- iting partial skin graft loss at the
site at which the fascia ap was inset were treated via secondary skin grafts. The
remaining 19 cases did not experience vascular or recipient site complications. In our
study, ow-through ALT aps were designed in nine cases that involved major
artery damage. The limbs of eight of these cases were successfully salvaged; the only
case of limb loss involved a 54- year-old man who was the victim of a trafc accident,
and this case involved degloving injuries and a completely severed left forearm.
Replantation was performed, and a ow-through ALT perforator ap was transferred
to reconstruct the radial artery and to cover the massive soft tissue loss. On
postoperative day 6, the distal left forearm became necrotic, and the patient
underwent amputation. However, the ALT ap survived and was used to cover the
stump.
Over the 18 monthse30 months (mean, 24 months) of follow-up in this study, the
aps remained viable, and the wounds healed well, including reliable soft tissue and
good contour in the recon- structed areas. Skin grafts over the fascia or muscle
layers devel- oped very well (i.e., smoothly with great elasticity, no recoil, and no
change in color). Three patients received subsequent ap debulk- ing. All of the
donor sites were closed using split-thickness skin grafts. Among the 22 patients,
there were no complications at the donor sites that required surgical treatment. No
patients experi- enced any difculty in activities of daily living, and none of the
patients suffered from knee extension lag. No patients experienced restrictions in
climbing stairs. Only six patients experienced transient limb weakness. These six
patients typically regained their preoperative range of mobility and activity level
within six months. The appearance of the donor site was considered to be satisfactory
by 18 patients and acceptable by four patients.

3.1. Case report

3.1.1. Case 1
A 20-year-old man involved in a trafc accident sustained an injury to the right
lower limb. He presented with open ankle joint dislocation and irregular and
extensive soft tissue defects measuring 43
35 cm on the distal leg (Fig. 2A, B). In
addition, both the anterior and posterior tibial arteries were ruptured, resulting in
impaired distal leg and foot circulation.
Two teams worked together to perform the operation. The ankle joint dislocation
was reduced and xed using pins. Subsequently, the ruptured posterior tibial artery
was repaired using a great saphenous vein graft to restore the blood supply. Based
on the size and characteristics of the wounds, a chimeric ALT perforator ap was
harvested that consisted of a skin component, a partial vastus lateralis muscle
component, and a fascial pedicle on the left descending branch of the LCFA (Fig. 2C).
The majority of the vastus lateralis muscle remained in situ and functionally intact.
The skin paddle was 32
13 cm; the fascia ap was 15
10 cm; and the muscle
ap was 10
8 cm. The skin ap was applied to the ante- romedial surface of the leg
to cover the underlying gliding tissues and exposed tendons. The muscle ap
covered the posterior sur- face, whereas the fascia ap covered the lateral surface

28
(Fig. 2D, E). The pedicle vessels and the descending branch were interposed between
the two ends of the anterior tibial artery, which exhibited a 3-cm gap. The muscle
and fascia aps were covered by a skin graft.
The donor site was closed using a skin graft. The ap survived in its entirety; partial skin
graft loss was treated via a secondary skin graft. The patient was not satised with the
appearance of the aps and received secondary ap debulking after 5 months. The
nal follow-up occurred at 18 months (Fig. 2F). The functional recovery of the lower
limb was satisfactory.

3.1.2. Case 2
A 20-year-old man sustained an
incompletely severed right ankle with an
extensive soft tissue defect as a result of a trafc accident. A 30
24 cm soft
tissue defect was present after debridement, open reduction, and internal xation
(Fig. 3A, B). The posterior tibial artery was immediately repaired using a vein graft. A
large free chimeric ALT ap consisting of a skin ap (18
12 cm), a fascia ap (12
10
cm), and a muscle ap (8 6 cm) was har- vested with its associated perforators (Fig.
3C). Based on the size and characteristics of the wound, the skin ap covered the
ante- rolateral surface of the wound; the fascia ap covered the medial surface of the
wound; and the muscle ap covered the posterior surface of the wound (Fig.
3D, E). Vascular ow-through anastomosis was performed on the anterior tibial
vessels. The defatted glabrous skin was repositioned using a full-sheet skin graft.
The ipsilateral donor site was grafted using a split-thickness skin graft. The ap
survived in its entirety without any major compli- cations (Fig. 3F). Partial skin graft
loss occurred at the sites at which the fascia ap was inset; this loss was treated via a
secondary skin graft. The patient recovered partial motion of the ankle joint.

4. Discussion
Large and complex traumatic defects in the extremity remain a challenge for surgeons
with restoration of function with minimal donor-site morbidity being the surgical
goals. Traditionally, latis- simus dorsi, thoracodorsal ap and rectus abdominis muscle
ap have been used for the reconstruction of larger defects [6]. Compared with
the thoracic or trunk areas as donor site, an often- cited benet of the ALT ap is the
minimal loss of function and the cosmetically acceptable donor site,
which is
covered by normal clothing [7]. In our department, the ALT ap is extensively used for
the reconstruction of complex defects of the upper or lower ex- tremities. However,
the repair of extensive, irregular, ring-like soft tissue defects or degloving injuries of
the extremities using classic ALT aps is difcult. Based on the recently developed
perforator ap concept, if more than one perforator is available, the ALT ap can be
separated into several different tissue aps; each ap is based on a single perforator.
This ALT ap design, referred to as a compound ap or a chimeric perforator ap, is a
modication of the classic ALT ap in which a small area of skin is harvested at the
donor site to yield the largest amount of tissue possible for the reconstruction of
extensive, irregular, and complex defects via a single vascular anastomosis while
inducing minimal loss of func- tion [8,9]. And this chimeric ALT ap is versatile for
the recon- struction of three-dimensional defects in the extremities. Because different

29
tissue aps contain perforators that are independent from the main trunk of the
descending branch of the LCFA, it is easy to cover extensive and irregular wounds
freely in various directions; thus, complex three-dimensional defects can
be
reconstructed. Based on chimeric ap and perforator dissection technique, Peng et al.
described their experience with the use
of chimeric ALT perforator aps to
simultaneously reconstruct two separate defects in the upper extremity [5]. Chou et
al. reported that two fas- ciocutaneous aps associated with independent skin
vessels could be reliably harvested from the same descending branch of the LCFA for
the simultaneous reconstruction of two separate defects [10]. Both groups split the
skin paddle between the perforators to generate two separate paddles with a
common vascular supply. Jiang et al. also reported one tripaddled ALT ap for
reconstruction of bilateral buccal defects created from cancer ablation and severe
contralateral oral submucous brosis release [11]. However, few reports are
available that describe the simultaneous reconstruction of more extensive, complex,
and irregular defects using three different tissue aps from the same descending
branch of the LCFA. The design of ap allowed the three or two independent aps
easier to inset for reconstruction of complex defects.
In this report, we present the results of the use of chimeric ALT perforator aps, which
enabled the development of two or three independent free tissue aps based on the
same descending branch of the LCFA. This chimeric ALT perforator ap was
designed for extremity reconstruction, considering the size and characteristics of the
soft tissue defects and the location and characteristics of the recipient sites [12]. For
large defect areas, we primarily used the skin ap to cover sites containing nerves or
vessels. For small defect areas or less important sites, we primarily used fascia aps
and subsequently covered the site with a split skin graft. In cases of overly large
defects for which the two aforementioned aps were insufcient or in cases of cavity
or bone exposure repair, bone de- fects, or severe contamination, we performed the
reconstruction using an additional muscle ap. Muscle tissue is highly vascular and,
thus, is highly resistant to infection [1,13,14]. In some cases of severe injury to the
extremities, deep defects may be associated with major arterial damage, leading to
poor perfusion of the distal region of the limb. Flow-through aps appear to represent
the best option in these situations because such aps enable simultaneous arterial
reconstruction and soft-tissue coverage [15]. The chimeric ALT perforator ap can be
utilized for extremity reconstruction as a ow-through ap in which the pedicle
bridges the vascular gaps [16]. In our study, ow-through ALT perforator aps were
designed in 9 cases. Vascular ow-through anastomosis was performed on the main
trunk of the vessels to provide simultaneous arterial reconstruction and soft-tissue
coverage of the extremity. In the majority of cases, the muscle, fascia, and skin aps
were generated completely independently by dissecting muscle, fascia, and skin on
separate, individual perforators. In the vastus lateralis muscle, most of the large muscle
branches emanating from the myocutaneous perforators run medially, laterally, and
posteriorly from the descending branch, and ap harvesting can be completed via the
standard method [17]. In some cases, the main pedicle associated with the fascia ap
originated from the perforators of the skin ap and did not directly originate from the
descending branch of the LCFA. In some cases, there were many tiny perforators that
could not be freely dissected with the fascia ap; in these cases, a pedicle that was
2e3 cm wide was preserved to ensure the circulation of the ap. In the latter two
types of aps, a similar freedom of rota- tion could still be achieved when the skin and

30
muscle islands were isolated on an individual perforator. Before surgery, we routinely
performed assessment via hand-held Doppler owmetry to reduce the uncertainty
caused by variations in the vascular anatomy. The authors with the most experience
in this procedure recommended this procedure [18,19]. The localization and course of
the perfora- tors could be determined accurately, and vascular anomalies could be
identied [20]. During surgery, all of the perforators from the descending branch of
the LCFA should be identied and preserved. We suggest that these complex chimeric
aps are ideally dissected from the pedicle to the various tissue components of the
chimeric ap.
The
initial dissection of the vascular pedicle facilitates the
identication of any vascular anomalies and the assessment of the quantity and
quality of the perforators while the extirpative sur- geon is completing the resection
[21].
This type of chimeric ALT perforator aps provides several benets. First, a minimal
area of skin is harvested at the donor site to provide the largest amount of tissue
possible for the coverage of a massive soft tissue defect with minimal loss of function
and good cosmetic acceptability at the donor site. Second, the various pad- dles can
be inset with many degrees of freedom, facilitating the coverage of complex and
extensive three-dimensional defects in the extremities. Third, the various tissue
components can be used for the reconstruction of complex extremity defects that
include multiple tissue structural requirements. Finally, this ap can be designed
with minimal effort and operation time as well as one pair of recipient vessels [22]. A
disadvantage of the chimeric ALT perforator ap technique is that it requires
advanced skill. The course of the perforators can be unpredictable, and small or long
perforators can be difcult to harvest and inset. The microsurgeon must have superior
microsurgical skills and must be familiar with perforator aps and intramuscular
perforator dissection [23]. Finally,
for such extensive and complex soft
tissue
defects, we suggest a two-team surgery, during which the teams performing
resection and reconstruction operate in parallel to reduce the operation time.
Our study is subject to the limitations inherent in retrospective, uncontrolled case
reviews, including a small sample size, no strict criteria for patient selection, and no
prospective data collection, especially a nal strict functional assessment.
5. Conclusions
In summary, the chimeric ALT perforator ap, which is a novel variation of the
standard ALT ap design, was found to provide large tissue components that are
versatile and valuable. This technique facilitates the harvest of various tissue
components with maximal freedom, providing maximal exibility to meet specic
reconstructive requirements for large, complex, and irregular soft- tissue defects in
the extremities.

Ethical approval
All studies have been approved by both the First Afliated Hospital of Sun Yatsen University and Xian Fengcheng Hospital Ethics Committee and performed in
accordance with the ethical standards.

31
Author contribution

Canbin Zheng participated in the design of the operation and study, and drafted
the manuscript. Xiaoju Zheng carried out the operation, together with Canbin
Zheng and helped to draft the manuscript. Baoshan Wang took charge of collecting
the patients data and helped to perform the operation. Yongfeng Qiu took charge
of recruiting patients and helped to design the study. Zhong Zhang and Haijun Li
followed up all the patients and analyzed the data. Xinhong Wang collected
important background information. All authors read and approved the nal
manuscript.