Quantitative assessment of graded burn wounds using a commercial and research grade laser speckle imaging (LSI) system

Burn wounds are often characterized by injury depth, which then dictates wound management strategy. While most superficial burns and full thickness burns can be diagnosed through visual inspection, clinicians experience difficulty with accurate diagnosis of burns that fall between these extremes. Accurately diagnosing burn severity in a timely manner is critical for starting the appropriate treatment plan at the earliest time points to improve patient outcomes. To address this challenge, research groups have studied the use of commercial laser Doppler imaging (LDI) systems to provide objective characterization of burn-wound severity. Despite initial promising findings, LDI systems are not commonplace in part due to long acquisition times that can suffer from artifacts in moving patients. Commercial LDI systems are being phased out in favor of laser speckle imaging (LSI) systems that can provide similar information with faster acquisition speeds. To better understand the accuracy and usefulness of commercial LSI systems in burn-oriented research, we studied the performance of a commercial LSI system in three different sample systems and compared its results to a research-grade LSI system in the same environments. The first sample system involved laboratory measurements of intralipid (1%) flowing through a tissue simulating phantom, the second preclinical measurements in a controlled burn study in which wounds of graded severity were created on a Yorkshire pig, and the third clinical measurements involving a small sample of clinical patients. In addition to the commercial LSI system, a research grade LSI system that was designed and fabricated in our labs was used to quantitatively compare the performance of both systems and also to better understand the “Perfusion Unit” output of commercial systems.


INTRODUCTION
On an annual basis in the United States, 500,000 will require treatment for a burn injury and almost 10% of that number will be admitted into a specialized burn center [1].The most difficult task for physicians in these burn units is to accurately categorize the burn injury.Accurate stratification is important because the treatment plan for a patient is directly related to the initial diagnosis.A burn that is fairly superficial will not require any surgical intervention.A full thickness burn where significant damage is done requires surgical intervention.It is not difficult for clinicians to categorize burn wounds in their extreme state, but it is difficult to categorize the partial thickness burns that are in between the extreme categories.The accuracy of diagnosing these partial thickness burns by clinicians is typically 60-80% when the diagnosis is done several days after the burn injury [2].While early intervention has been shown to be beneficial for burn patients, clinical assessments are more accurate if clinicians wait to make a diagnosis [2].New technologies that could aid clinicians in categorizing wound severity shortly after injury would provide great benefit to patients.
Recently we reviewed technologies that have been employed to assess burn severity [3].One of the technologies that have found some acceptance in burn centers and appears to offer the best data-supported estimates of burn severity to date is Laser Doppler Imaging (LDI).LDI allows a clinician to visualize spatial maps of perfusion, a key indicator of burn depths and eventual healing times.One of the issues associated with LDI is long acquisition times because the laser is mechanically scanned over the patient for up to 5 minutes per image, resulting in a considerable risk of movement artifact and long data collection times for burns that cover a large area.An alternative technology under study is Laser speckle imaging (LSI), which is a similar noninvasive technique that also quantifies blood flow by examining the fluctuations in a speckle pattern caused by the movement of red blood cells.While LDI and LSI both report measurements of blood flow, LSI has the ability to capture an entire field of view at a single time point and can avoid artifacts due to movements.
To better understand the accuracy and usefulness of commercial LSI systems in burn-oriented research, we studied the performance of a commercial LSI system in three different environments and compared its results to a research-grade LSI system [4] in the same sample systems.The first sample system involved laboratory measurements of intralipid (1%) flowing through a tissue simulating phantom.The second system involved preclinical measurements in a controlled burn study in which wounds of graded severity were created on a Yorkshire pig (UCI IACUC # 2015-3154-0).Finally, we look at performance in a clinical setting involving a small sample of patients (UCI IRB #2009-7322).

Commercial laser speckle imaging system
The PeriCam PSI (Perimed AB, Sweden) is an imager that utilizes laser speckle contrast analysis to provide real time analysis of tissue perfusion.It utilizes a single-exposure speckle algorithm with background correction and is calibrated with a Brownian motion standard.Speckle contrast values are calculated at each pixel using the equation below: Where <I> is the mean intensity around a square area of pixels and σ(Ι) is the standard deviation of the intensity around those pixels.This speckle contrast value is converted to a "perfusion unit" measurement that is recorded and displayed to the end user using the equation below.
Where G is a gain factor reported by the software.

Research grade laser speckle imaging system
The research-grade LSI instrument, which was developed at BLI, consisted of three main hardware components: a laser source, CCD camera, and computer.The laser source was a continuous-wave laser light (λ = 808 nm, 150 mW, Ondax Inc., Monrovia, CA).The CCD camera was a thermoelectrically-cooled Retiga 2000R (QImaging, Burnaby, BC, Canada) with a pixel resolution of 1600 x 1200 (1.92 megapixels, each pixel having dimensions of 7.4 µm x 7.4 µm).
The laptop (Sager P170EM, City of Industry, CA) was equipped with a GTX650 Graphics Processing Unit (GPU) (NVIDIA, Santa Clara, CA).The speckle pattern generated by the laser was captured with the CCD camera and processed using custom LabVIEW (Version 8.0, National Instruments, Austin, TX) software, integrating CUDA GPU code to perform real time processing as described previously [5].Blood flow is quantified by a metric known as Speckle Flow Index (SFI), which we have described previously [6,7] using the equation below: Where T is the exposure time of the camera and K is the speckle contrast value.

Flow Phantoms
A flow phantom designed to approximate human tissue was constructed out of silicone (PDMS) using India ink as the absorbing agent and titanium dioxide as the scattering agent.An acrylic tube with dimensions 13 mm x 13 mm was embedded in the phantom to act as a flow channel.The tube was approximately 500 μm beneath the surface of the silicone.A syringe pump (NE-1000, New Era Pump Systems, Farmingdale, NY) was connected to the tubing and 1% intralipid was pumped through the setup.The pumping speed was increased from 0 -5 mm/s at increments of 1 mm/s with a 60s wait time in between increases.This range was chosen to approximate a range of red blood cell velocities encountered in microcirculation [8].

Porcine Studies
All experiments were performed in accordance with the UC Irvine Institutional Animal Care and Use Committee (IACUC protocol #2015-3154).Three Yorkshire pigs (55 kg average weight) were used in this experiment.Animals were allowed to acclimate to their individual housing for 7 days prior to the experiment.An intramuscular injection of tiletamine-zolazepam (Telazol, 6 mg/kg) was used to sedate the animals before being intubated so that isoflurane (2%) could be used to maintain sedation.Controlled, graded burn wounds were created using a custom-made device setup described previously [9].Briefly, custom made cylindrical brass probes (3 cm diameter) with stainless steel posts were fabricated along with a spring loaded device to hold the probes.The brass probes were heated in a warm bath incubator to 100°C and held in place by a custom made aluminum block.16 burns were created along the dorsal side approximately 1 cm from the spine and approximately 3 cm away from each other.8 wounds were created on each side of the spine, with contact times of 5, 10, 15, 20, 25, 30, 35, and 40 seconds.A non-adherent gauze (Telfa, Tyco Healthcare, Mansfield, MA) was placed on top of the wound and held in place with Ioban™ (3M, St. Paul, MN).

Preliminary Clinical Imaging
University of California Irvine (UCI) IRB approved protocol (HS #2009-7322) was used to enroll nine subjects into the study.Subjects were recruited from patients seen in the burn and surgical intensive care unit at UCI Medical Center by the attending physician (N.B.).Subjects who agreed to participate signed an informed consent form.Once informed consent was obtained, target thermal burn wounds of indeterminate depth were selected and imaged using the Pericam PSI system.Dressings, if present, were removed and standard wound care was provided by the health care provider.The physician remained blinded to the perfusion images to prevent any potential bias during visual assessment.Patients were checked for progress of treatments typically ten days after their admitted date.

Statistical Analysis
For flow phantom measurements a 1 cm x 5 cm region of interest was selected within the boundary of the flow channel and relative perfusion values at different speeds were calculated from baseline values for both systems.A paired t-test was performed on the two sets of data, with a 95% confidence interval.A p-value less than 0.05 was considered statistically significant for this study.Sensitivity measurements were calculated for each system using the equation below: Where P k is the measured perfusion at a given speed k.
For the porcine studies a 1 cm 2 ROI was selected within each burned section for both systems.The perfusion values were averaged across burns of the same severity over all animals.A paired t-test was performed on the two sets of data, with a 95% confidence interval.A p-value less than 0.05 was considered statistically significant for this study.
For the clinical measurements a 1 cm 2 ROI selection was chosen based on areas of interest pointed out by the attending physician.The perfusion values were then averaged within each ROI and compared.

Flow Phantoms
The PeriCam PSI and research-grade LSI system use separate units to express perfusion, so relative flow measurments were used to associate the two.The relative flow measurements for each system were determined by referencing the flow at each speed to the baseline measurement of 0 mm/s, when intralipid is stagnant within the flow phantom as illustrated in Fig 1 .Comparing the trend of this percent change of a flow measurement from baseline indicated that both systems registered an increase in perfusion as the pump increased flow.Overall, the PeriCam PSI system expressed a higher percent change from baseline than the research-grade system, for each of the measured flow speeds.The sensitivity of each system to varying speeds was determined by averaging the percent change between each flow speed for all five measured speeds.The sensitivity for the PeriCam PSI system was 0.23, whereas the sensitivity for the research grade LSI system was 0.17.

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