Leonid Zubkov

Image analysis of chronic wounds for determining the surface area

Progress in wound healing is primarily quantified by the rate of change of the wounds surface area. The most recent guidelines of the Wound Healing Society suggest that a reduction in wound size of <40% within 4 weeks necessitates a reevaluation of the treatment. However, accurate measurement of wound size is challenging due to the complexity of a chronic wound, the variable lighting conditions of examination rooms, and the time constraints of a busy clinical practice. In this paper, we present our methodology to quantify a wound boundary and measure the enclosed wound area reproducibly. The method derives from a combination of color‐based image analysis algorithms, and our results are validated with wounds in animal models and human wounds of diverse patients. Images were taken by an inexpensive digital camera under variable lighting conditions. Approximately 100 patient images and 50 animal images were analyzed and a high overlap was achieved between the manual tracings and the calculated wound area by our method in both groups. The simplicity of our method combined with its robustness suggests that it can be a valuable tool in clinical wound evaluations. The basic challenge of our method is in deep wounds with very small surface areas where color‐based detection can lead to erroneous results and which could be overcome by texture‐based detection methods. The authors are willing to provide the developed MATLAB code for the work discussed in this paper.

Correlation of near infrared absorption and diffuse reflectance spectroscopy scattering with tissue neovascularization and collagen concentration in a diabetic rat wound healing model

The objective of this paper was to correlate optical changes of tissue during wound healing measured by near infrared (NIR) and diffuse reflectance spectroscopy (DRS) with histologic changes in an animal model. Amplitude and phase of scattered light were obtained in a diabetic rat and control model and biopsies were taken for blood vessel ingrowth and collagen concentration. NIR absorption coefficient correlated with blood vessel ingrowth over time, in both the control and diabetic animals. DRS data correlated with collagen concentration. Previous publications by this group documented only the NIR changes during the wound healing process but this is the first reported correlation with histology data. The ability to correlate DRS scattering with collagen concentration during healing is another important and novel finding. This technology may play an important role clinically in assessing the efficacy of wound healing agents in diabetics.

Optical properties of wounds: diabetic versus healthy tissue

Diffuse photon density wave (DPDW) methodology at Near Infrared frequencies has been used to calculate absorption and scattering from wounds of healthy and diabetic rats. The diffusion equation for semi-infinite media is being used for calculating the absorption and scattering coefficients based on measurements of phase and amplitude with a frequency domain device. Differences observed during the course of healing in the two populations can be correlated to the delayed healing observed in diabetics. These results are encouraging and further work will focus on the implementation of this device to the clinical setting as a monitoring tool in chronic diabetic wounds.

Noninvasive assessment of diabetic foot ulcers with diffuse photon density wave methodology: pilot human study

A pilot human study is conducted to evaluate the potential of using diffuse photon density wave (DPDW) methodology at near-infrared (NIR) wavelengths (685 to 830 nm) to monitor changes in tissue hemoglobin concentration in diabetic foot ulcers. Hemoglobin concentration is measured by DPDW in 12 human wounds for a period ranging from 10 to 61 weeks. In all wounds that healed completely, gradual decreases in optical absorption coefficient, oxygenated hemoglobin concentration, and total hemoglobin concentration are observed between the first and last measurements. In nonhealing wounds, the rates of change of these properties are nearly zero or slightly positive, and a statistically significant difference (p<0.05) is observed in the rates of change between healing and nonhealing wounds. Differences in the variability of DPDW measurements over time are observed between healing and nonhealing wounds, and this variance may also be a useful indicator of nonhealing wounds. Our results demonstrate that DPDW methodology with a frequency domain NIR device can differentiate healing from nonhealing diabetic foot ulcers, and indicate that it may have clinical utility in the evaluation of wound healing potential.

Prediction of wound healing in human diabetic foot ulcers by diffuse near‐infrared spectroscopy: A pilot study

A human study was conducted in which the efficacy of in vivo diffuse near‐infrared (NIR) spectroscopy was demonstrated in predicting wound healing in diabetic foot ulcers. Sixteen chronic diabetic wounds were followed and assessed for subsurface oxy‐hemoglobin concentration using the NIR device. Weekly measurements were conducted until there was wound closure, limb amputation, or 20 completed visits without healing. Digital photography measured wound size, and the degree of wound contraction was compared with the NIR results. In the 16 patients followed, seven wounds healed, six limbs were amputated, and three wounds remained opened after 20 visits. The initial values in subsurface hemoglobin concentration in all wounds were higher than the nonwound control sites. Healed wounds showed a consistent reduction of hemoglobin concentration several weeks before closure that approached control site values. In wounds that did not heal or resulted in amputation of the limb, the hemoglobin concentration remained elevated. In some cases, these nonhealing wounds appeared to be improving clinically. A negative slope for the rate of change of hemoglobin concentration was indicative of healing across all wounds. In conclusion, evaluation of wounds using NIR may provide an effective measurement of wound healing. NIR spectroscopy can determine wound healing earlier than that visibly assessed by current clinical approaches.

Changes in optical properties of tissue during acute wound healing in an animal model

Changes of optical properties of wound tissue in hairless rats were quantified by diffuse photon density wave methodology at near-infrared frequencies. The diffusion equation for semi-infinite media was used to calculate the absorption and scattering coefficients based on measurements of phase and amplitude with a frequency domain device. There was an increase in the absorption and scattering coefficients and a decrease in blood saturation of the wounds compared with the nonwounded sites. The changes correlated with the healing stage of the wound. The data obtained were supported by immunohistochemical analysis of wound tissue. These results verified now by two independent animal studies could suggest a noninvasive method to detect the progress of wound healing.

Low-frequency (<100 kHz), low-intensity (<100 mW/cm2) ultrasound to treat venous ulcers: A human study and in vitro experiments

The purpose of this study was to examine whether low frequency (<100 kHz), low intensity (<100 mW/cm(2), spatial peak temporal peak) ultrasound can be an effective treatment of venous stasis ulcers, which affect 500 000 patients annually costing over

Optimization of un-tethered, low voltage, 20-100kHz flexural transducers for biomedical ultrasonics applications.

1 billion per year. Twenty subjects were treated with either 20 or 100 kHz ultrasound for between 15 and 45 min per session for a maximum of four treatments. Healing was monitored by changes in wound area. Additionally, two in vitro studies were conducted using fibroblasts exposed to 20 kHz ultrasound to confirm the ultrasounds effects on proliferation and cellular metabolism. Subjects receiving 20 kHz ultrasound for 15 min showed statistically faster (p < 0.03) rate of wound closure. All five of these subjects fully healed by the fourth treatment session. The in vitro results indicated that 20 kHz ultrasound at 100 mW/cm(2) caused an average of 32% increased metabolism (p < 0.05) and 40% increased cell proliferation (p < 0.01) after 24 h when compared to the control, non-treated cells. Although statistically limited, this work supports the notion that low-intensity, low-frequency ultrasound is beneficial for treating venous ulcers.

A PMMA microcapillary quantum dot linked immunosorbent assay (QLISA)

This paper describes optimization of un-tethered, low voltage, 20-100kHz flexural transducers for biomedical ultrasonics applications. The goal of this work was to design a fully wearable, low weight (<100g), battery operated, piezoelectric ultrasound applicator providing maximum output pressure amplitude at the minimum excitation voltage. Such implementation of ultrasound applicators that can operate at the excitation voltages on the order of only 10-25V is needed in view of the emerging evidence that spatial-peak temporal-peak ultrasound intensity (I(SPTP)) on the order of 100mW/cm(2) delivered at frequencies below 100kHz can have beneficial therapeutic effects. The beneficial therapeutic applications include wound management of chronic ulcers and non-invasive transdermal delivery of insulin and liposome encapsulated drugs. The early prototypes of the 20 and 100kHz applicators were optimized using the maximum electrical power transfer theorem, which required a punctilious analysis of the complex impedance of the piezoelectric disks mounted in appropriately shaped metal housings. In the implementation tested, the optimized ultrasound transducer applicators were driven by portable, customized electronics, which controlled the excitation voltage amplitude and facilitated operation in continuous wave (CW) or pulsed mode with adjustable (10-90%) duty cycle. The driver unit was powered by remotely located rechargeable lithium (Li) polymer batteries. This was done to further minimize the weight of the applicator unit making it wearable. With DC voltage of approximately 15V the prototypes were capable of delivering pressure amplitudes of about 55kPa or 100mW/cm(2) (I(SPTP)). This level of acoustic output was chosen as it is considered safe and side effects free, even at prolonged exposure.

Measurement of optical properties to quantify healing of chronic diabetic wounds

The development of a simple and inexpensive quantum dot based immunoassay for detecting myeloperoxidase (MPO) in stool samples is reported (QLISA). The method developed utilizes readily available polymethylmethacrylate (PMMA) microcapillaries as substrates for performing the sandwich assay. High power (80 mW) and low power (10 mW) UV-LEDs were tested for their efficiency in maximizing detection sensitivity in a waveguide illumination or a side illumination mode. The results obtained indicate that both waveguide and side illumination modes can be employed for detecting MPO down to 15 ng/mL, however the high power LED in a side illumination mode improves sensitivity and simplifies the data acquisition process. The protocol and sensor robustness was evaluated with animal stool samples spiked with MPO and the results indicate that the sensitivity of detection is not compromised when used in stool samples. The effect of the ionic strength of the environment on the fluorescence stability of quantum dots was evaluated and found to affect the assay only if long imaging times are employed. Replacing the buffer with glycerol during imaging increased the fluorescence intensity of quantum dots while significantly minimized the loss in intensity even after 2h.