Sources of variability in fluorescence spectroscopic measurements in a Phase II clinical trial of 850 patients.

BACKGROUND: Devices using fluorescence spectroscopy to differentiate high grade squamous intraepithelial lesions from normal tissue in the cervix have shown some diagnostic efficacy. Measurements from these devices produce large amounts of complex, multi-factored data. The purpose of this study is to isolate the effects of the particular care providers and equipment operators who are involved in taking measurements. METHODS: Data from spectroscopic measurements of the Phase II study of 850 patients with abnormal Papancicolau smears were used. The data were subject to a Principal Components Analysis and to MANOVA to control for variables that are known to affect outcomes. RESULTS: The analysis showed significant provider effects from both devices and significant operator effects from one device. CONCLUSION: We will repeat a similar analysis on data from a screening trial, on the combined diagnostic and screening study, and on the reflectance data. In the future, we hope to be able to design devices that address provider and operator effects.

Methodology of a real-time quality control for the multispectral digital colposcope (MDC).

BACKGROUND: The diagnostic ability of algorithms developed for the Multispectral Digital Colposcope (MDC) is highly dependent on the quality of the image. The field of objective medical image quality analysis has great potential but has not been well exploited. Various researchers have reported different measures of image quality but with an existence of a reference image. The quality of an image can be attributed to several sources of errors, a few of which would be inclusion of presence of extraneous components, improper illumination, or an image out of focus. This can be due to motion artifact or the region of interest out of the focal plane. METHODS: With spectroscopic measurements, assessment of data quality has been used by our group in the past to avoid hardware errors at the time of acquisition. We are currently developing algorithms that will help identify hardware and acquisition errors to the clinician in under a few seconds. RESULTS: Minimizing these errors not only provides quality images for a diagnostic algorithm, but reduces the necessity for complex and time intensive post-processing software for enhancing the images. CONCLUSION: We propose a no reference image quality system specifically designed for MDC that can be modified to similar spectroscopic imaging applications.

Fluorescence and reflectance device variability throughout the progression of a phase II clinical trial to detect and screen for cervical neoplasia using a fiber optic probe.

Large phase II trials of fluorescence and reflectance spectroscopy using a fiber optic probe in the screening and diagnostic settings for detecting cervical neoplasia have been conducted. We present accrual and histopathology data, instrumentation, data processing, and the preliminary results of interdevice consistencies throughout the progression of a trial. Patients were recruited for either a screening trial (no history of abnormal Papanicolaou smears) or a diagnostic trial (a history of abnormal Papanicolaou smears). Colposcopy identified normal and abnormal squamous, columnar, and transformation zone areas that were subsequently measured with the fiber probe and biopsied. In the course of the clinical trial, two generations of spectrometers (FastEEM2 and FastEEM3) were designed and utilized as optical instrumentation for in vivo spectroscopic fluorescence and reflectance measurements. Data processing of fluorescence and reflectance data is explained in detail and a preliminary analysis of the variability across each device and probe combination is explored. One thousand patients were recruited in the screening trial and 850 patients were recruited in the diagnostic trial. Three clinical sites attracted a diverse range of patients of different ages, ethnicities, and menopausal status. The fully processed results clearly show that consistencies exist across all device and probe combinations throughout the diagnostic trial. Based on the stratification of the data, the results also show identifiable differences in mean intensity between normal and high-grade tissue diagnosis, pre- and postmenopausal status, and squamous and columnar tissue type. The mean intensity values of stratified data show consistent separation across each of the device and probe combinations. By analyzing trial spectra, we provide more evidence that biographical variables such as menopausal status as well as tissue type and diagnosis significantly affect the data. Understanding these effects will lead to better modeling parameters when analyzing the performance of fluorescence and reflectance spectroscopy.

Instrumentation as a source of variability in the application of fluorescence spectroscopic devices for detecting cervical neoplasia.

We report on a study designed to assess variability among three different fluorescence spectroscopy devices, four fiber optic probes, and three sets of optical calibration standards to better understand the reproducibility of measurements and interdevice comparisons of fluorescence spectroscopic data intended for clinical diagnostic use. Multiple measurements are acquired from all sets of standards using each combination of spectrometer, fiber optic probe, and optical standard. Data are processed using standard calibration methods to remove instrument-dependant responses. Processed spectra are analyzed using an analysis of variance to assess the percent variance explained by each factor that was statistically significant. Analysis of processed data confirms statistically significant differences among the spectrometers and fiber optic probes. However, no differences are found when varying calibration standards or measurement date and time. The spectrometers and fiber optic probes are significant sources of variability, but appropriate data processing substantially reduces these effects. Studies of inter- and intradevice variability are important methodological issues for optical device trials and must be included in the quality assurance studies for the clinical trial design.

Laser-induced collagen remodeling and deposition within the basilar membrane of the mouse cochlea.

The cochlea is the mammalian organ of hearing. Its predominant vibratory element, the basilar membrane, is tonotopically tuned, based on the spatial variation of its mass and stiffness. The constituent collagen fibers of the basilar membrane affect its stiffness. Laser irradiation can induce collagen remodeling and deposition in various tissues. We tested whether similar effects could be induced within the basilar membrane. Trypan blue was perfused into the scala tympani of anesthetized mice to stain the basilar membrane. We then irradiated the cochleas with a 694-nm pulsed ruby laser at 15 or 180 Jcm(2). The mice were sacrificed 14 to 16 days later and collagen organization was studied. Polarization microscopy revealed that laser irradiation increased the birefringence within the basilar membrane in a dose-dependent manner. Electron microscopy demonstrated an increase in the density of collagen fibers and the deposition of new fibrils between collagen fibers after laser irradiation. As an assessment of hearing, auditory brainstem response (ABR) thresholds were found to increase moderately after 15 Jcm(2) and substantially after 180 Jcm(2). Our results demonstrate that collagen remodeling and new collagen deposition occurs within the basilar membrane after laser irradiation in a similar fashion to that found in other tissues.

Calibration standards for multicenter clinical trials of fluorescence spectroscopy for in vivo diagnosis.

In the context of clinical trials, calibration protocols for optical instruments that ensure measurement accuracy and the ability to carry out meaningful comparisons of data acquired from multiple instruments are required. A series of calibration standards and procedures are presented to assess technical feasibility of optical devices for cervical precancer detection. Measurements of positive and negative standards, and tissue are made with two generations of research grade spectrometers. Calibration accuracy, ability of standards to correct and account for changes in experimental conditions, and device components are analyzed. The relative frequency of measured calibration standards is investigated retrospectively using statistical analysis of trends in instrument performance. Fluorescence measurements of standards and tissue made with completely different spectrometers show good agreement in intensity and lineshape. Frequency of wavelength calibration standards is increased to every 2 h to compensate for thermal drifts in grating mount. Variations in illumination energy detected between standards and patient measurements require probe redesign to allow for simultaneous acquisition of illumination power with every patient measurement. The use of frequent and well-characterized standards enables meaningful comparison of data from multiple devices and unambiguous interpretation of experiments among the biomedical optics community.

Design and preliminary analysis of a study to assess intra-device and inter-device variability of fluorescence spectroscopy instruments for detecting cervical neoplasia.

INTRODUCTION: A study was designed to assess variability between different fluorescence spectroscopy devices. Measurements were made with all combinations of three devices, four probes, and three sets of standards trays. Additionally, we made three measurements on the same day over 2 days for the same combination of device, probe, and standards tray to assess reproducibility over a day and across days. MATERIALS AND METHODS: The devices consisted of light sources, fiber-optics, and cameras. We measured thirteen standards and present the data from the frosted cuvette, water, and rhodamine standards. A preliminary analysis was performed with the data that were wavelength calibrated and background subtracted; however, the system has not been corrected for systematic intensity variations caused by the devices. Two analyses were performed on the rhodamine, water, and frosted cuvette standards data. The first one is based on first clustering the measurements and then looking for association between the 5 factors (device, probe, standards tray, day, measurement number) using chi-squared tests on the cross-tabulation of cluster and factor level. This showed that only device and probe were significant. We then did an analysis of variance to assess the percent variance explained by each factor that was significant from the chi-squared analysis. RESULTS: The data were remarkably similar across the different combinations of factors. The analysis based on the clusters showed that sometimes devices alone, probes alone, but most often combinations of device and probe caused significant differences in measurements. The analysis showed that time of day, location of device, and standards trays do not vary significantly; whereas the devices and probes account for differences in measurement. We expected this type of significance using unprocessed data since the processing corrects for differences in devices. However, this analysis on raw data is useful to explore what combination of device and probe measurements should be targeted for further investigation. This experiment affirms that online quality control is necessary to obtain the best excitation-emission matrices from optical spectroscopy devices. CONCLUSION: The fact that the device and probe are the primary sources of variability indicates that proper correction for the transfer function of the individual devices should make the measurements essentially equivalent.

Laser-induced thermal injury to dermal blood vessels: analysis of wavelength (585 nm vs. 595 nm), cryogen spray cooling, and wound healing effects.

BACKGROUND AND OBJECTIVES: Successful laser treatment of cutaneous hyper-vascular lesions requires appropriate laser irradiation parameters for selective photothermolysis of ectatic dermal blood vessels as well as appropriate cooling parameters for epidermal protection based on an individual patient basis. Using the rabbit ear as an in vivo model for dermal vasculature, we investigated the influences of laser wavelength (585 nm vs. 595 nm) and cryogen spray cooling with various spurt durations on the laser-induced thermal injury to dermal blood vessels. Wound healing response was also evaluated in 2 hours and 4 days. STUDY DESIGN/MATERIALS AND METHODS: Flashlamp-pumped pulsed dye laser ScleroPlus (operated at the wavelength of 585 or 595 nm) was used for the comparison between the influences of two wavelengths (585 nm vs. 595 nm). R134-a cryogen spurts with the durations from 50 to 300 milliseconds were sprayed onto the sites to be irradiated and terminated 20 milliseconds before the onset of the laser pulses. In vivo rabbit ear was used as the model for cutaneous hyper-vascular lesions. Totally 10 New Zealand Albino white rabbits were experimented and in each rabbit ear six to seven sites were irradiated. Five animals were sacrificed 2 hours after the irradiation, and the remaining five sacrificed 4 days after the irradiation. Thermal injury to the blood vessel was assessed by hematoxylin and eosin stained histological sections and confirmed by an apoptosis assay. RESULTS: When the radiant exposures were above 10 J/cm2, 595 nm wavelength induced equivalent or more severe thermal injury to dermal blood vessels than 585 nm. Cryogen spray cooling with the spurt durations above 100 milliseconds resulted in increased depth of the most superficial thermal injury to dermal blood vessels than without cooling, indicating that superficial blood vessels were non-specifically cooled by the cryogen spurts applied at these parameters. Laser-induced thermal injury was significantly healed in the rabbit ear vasculature at 4 days post irradiation. CONCLUSIONS: Given sufficient radiant exposure, 595 nm wavelength can induce equivalent or more severe vascular injury compared with 585 nm. Cryogen spray cooling with the spurt durations above 100 ms may impair the photocoagulation of superficial blood vessels. Irreversible thermal injury to blood vessel can be achieved only when the basement membrane of blood vessel wall is irreversibly damaged.

Epidermal and vascular damage analysis of in vivo human skin in response to 595 nm pulsed laser irradiation.

BACKGROUND AND OBJECTIVES: Laser irradiation is the current modality for treatment of cutaneous hypervascular malformations such as port wine stains and telangiectasia. Although cryogen spray cooling (CSC) is used to protect the epidermis from non-specific laser-induced thermal damage in moderately-pigmented skin types, individuals with high melanin content are still at risk for epidermal damage using the current laser irradiation and CSC parameters. The objective of this study was to investigate the influence of the spray Weber number (1,100 or 5,100) on epidermal protection and examine vascular coagulation in response to pulsed dye laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: Normal, in vivo human skin from eight subjects of Fitzpatrick skin types I-V were precooled with either low or high Weber number cryogen sprays and subsequently irradiated with a pulsed dye laser at 595 nm. Analysis of gross purpura, morphological vascular damage, and apoptosis of the vascular walls were performed. RESULTS: Results demonstrated a high Weber number spray of 5,100 decreased the level of epidermal damage in darker and moderate pigmented individuals compared to a Weber number spray of 1,100. This study also established a positive correlation between gross purpura and the level of vessel wall apoptosis. CONCLUSIONS: This study has demonstrated that CSC with a high Weber number spray can decrease nonspecific thermal damage to the epidermis in response to laser irradiation in vivo. We have also established a positive correlation between gross purpura and the level of vessel wall apoptosis. Lasers Surg. Med. (c) 2005 Wiley-Liss, Inc.

Comparison of 585 and 595 nm laser-induced vascular response of normal in vivo human skin.

BACKGROUND AND OBJECTIVES: Two wavelengths, 585 and 595 nm, are currently common options for treating vascular malformations such as port-wine stains (PWS). Controversy exists as to which wavelength induces greater photothermal damage to the blood vessels and subsequent resolution of the malformations. STUDY DESIGN/MATERIALS AND METHODS: We irradiated normal, human skin in vivo at 585 and 595 nm wavelengths using fluences of 10-30 J/cm(2) with a 1.5 millisecond laser pulse. The level of purpura, total vascular damage, maximum coagulation depth (MCD), and perivascular damage were quantified by gross observation and histological analysis. RESULTS: Results demonstrated that 585 nm light caused greater purpura, vascular damage, maximum coagulation depth, and perivascular damage than 595 nm. Purpura showed a positive correlation with total vascular damage to a certain extent beyond which the total vascular damage did not change. For equivalent purpura, 585 and 595 nm produced no statistically significant difference in vascular damage. The difference in the laser-induced vascular damage between 585 and 595 nm, although statistically significant, was no more than 50%. CONCLUSIONS: The bathochromic (red) shift and formation of met-hemoglobin, which reduces the 585 nm light absorption and increases that of 595 nm compared to native oxy-hemoglobin, play a considerable role in creating more parity in vascular damage between the two wavelengths than would be expected based on their respective "native" absorption coefficients alone.

Comparison of human skin opto-thermal response to near-infrared and visible laser irradiations: a theoretical investigation.

Near-infrared wavelengths are absorbed less by epidermal melanin, and penetrate deeper into human skin dermis and blood than visible wavelengths. Therefore, laser irradiation using near-infrared wavelengths may improve the therapeutic outcome of cutaneous hyper-vascular malformations in moderately to heavily pigmented skin patients and those with large-sized blood vessels or blood vessels extending deeply into the skin. A mathematical model composed of a Monte Carlo algorithm to estimate the distribution of absorbed light, numerical solution of a bio-heat diffusion equation to calculate the transient temperature distribution, and a damage integral based on an empirical Arrhenius relationship to quantify the tissue damage was utilized to investigate the optothermal response of human skin to near-infrared and visible laser irradiations in conjunction with cryogen spray cooling. In addition, the thermal effects of a single continuous laser pulse and micropulse-composed laser pulse profiles were compared. Simulation results indicated that a 940 nm wavelength induces improved therapeutic outcome compared with a 585 and 595 nm wavelengths for the treatment of patients with large-sized blood vessels and moderately to heavily pigmented skin. On the other hand, a 585 nm wavelength shows the best efficacy in treating small-sized blood vessels, as characterized by the largest laser-induced blood vessel damage depth compared with 595 and 940 nm wavelengths. Dermal blood content has a considerable effect on the threshold incident dosage for epidermal damage, while the effect of blood vessel size is minimal. For the same macropulse duration and incident dosage, a micropulse-composed pulse profile results in higher peak temperature at the basal layer of skin epidermis than an ideal single continuous pulse profile.

Laser irradiation of the guinea pig basilar membrane.

BACKGROUND AND OBJECTIVES: The cochlea is the part of the inner ear that transduces sound waves into neural signals. The basilar membrane, a connective tissue sheet within the cochlea, is tonotopically tuned based on the spatial variation of its mass, stiffness, and damping. These biophysical properties are mainly defined by its constituent collagen fibers. We sought to assess the effect of laser irradiation on collagen within the basilar membrane using histological analysis. STUDY DESIGN/MATERIALS AND METHODS: Four excised guinea pig cochleae were stained with trypan blue. From these, two were irradiated with a 600 nm pulsed dye laser and two were used as controls. Collagen organization was visualized using polarization microscopy. RESULTS: Laser irradiation reduced the birefringence within the basilar membrane as well as within other stained collagen-containing structures. Larger reductions in birefringence were measured when more laser pulses were given. The effects were similar across all turns of each cochlea. CONCLUSIONS: Laser irradiation causes immediate alterations in collagen organization within the cochlea that can be visualized with polarization microscopy. These alterations may affect cochlear tuning. Ongoing research is aimed at analyzing the effect of laser irradiation on cochlear function. It is conceivable that this technique may have therapeutic benefits for patients with high-frequency sensorineural hearing loss.

Effects of droplet velocity, diameter, and film height on heat removal during cryogen spray cooling.

Cryogen spray cooling (CSC) is an effective method to reduce or eliminate epidermal damage during laser treatment of various dermatoses. This study sought to determine the effects of specific cryogen properties on heat removal. Heat removal was quantified using an algorithm that solved an inverse heat conduction problem from internal temperature measurements made within a skin phantom. A nondimensional parameter, the Weber number, characterized the combined effects of droplet velocity, diameter, and surface tension. CSC experiments with laser irradiation were conducted on ex vivo human skin samples to assess the effect of Weber number on epidermal protection. An empirical relationship between heat removal and the difference in droplet temperature and the substrate, droplet velocity, and diameter was obtained. Histological sections of irradiated ex vivo human skin demonstrated that sprays with higher Weber numbers increased epidermal protection. Results indicate that the cryogen film acts as an impediment to heat transfer between the impinging droplets and the substrate. This study offers the importance of Weber number in heat removal and epidermal protection.

Thermal response of human skin epidermis to 595-nm laser irradiation at high incident dosages and long pulse durations in conjunction with cryogen spray cooling: an ex-vivo study.

BACKGROUND AND OBJECTIVES: Improved laser treatment of cutaneous hypervascular lesions is expected by utilizing higher incident dosages, longer pulse durations and longer wavelengths than those currently used in clinical settings. However, simply increasing the incident dosage will also increase the risk of nonspecific thermal injury to the epidermis due to light absorption by melanin. In this study, we investigated the thermal response of human skin epidermis to 595-nm wavelength laser irradiation at high incident dosages (up to 20 J/cm(2)) and long pulse durations (up to 40 milliseconds) in conjunction with cryogen spray cooling (CSC) using ex-vivo human skin samples. STUDY DESIGN/MATERIALS AND METHODS: The Candela V-beam trade mark laser (595-nm wavelength) was used in the experiments. Ex-vivo human skin samples (Fitzpatrick types I-VI) were irradiated at the incident dosages D(0) = 4, 6, 10, 15, and 20 J/cm(2), laser pulse durations tau(laser) = 1.5, 10, and 40 milliseconds, without and with CSC (refrigerant-134A, spurt duration tau(CSC) = 100 milliseconds). Thermal injury to the epidermis was evaluated by histological observations. RESULTS: Under the same incident dosage, longer pulse durations led to reduced thermal injury to the epidermis. Without CSC, no demonstrable thermal injury to the epidermis was observed in skin types I-II irradiated at the incident dosage as high as 15 J/cm(2), and in skin types III-IV at 10 J/cm(2). When CSC was applied, no evidence of thermal injury to the epidermis was present in skin types I-II even when irradiated at the maximum available incident dosage of the laser system (20 J/cm(2)). In skin types III-IV, no demonstrable thermal injury to the epidermis was observed when using incident dosage as high as 15 J/cm(2) in conjunction with CSC. In skin type VI, thermal injury to the epidermis could not be avoided even at the setting D(0) = 4 J/cm(2), tau(laser) = 40 milliseconds in conjunction with CSC. CONCLUSIONS: For a given incident dosage, longer pulse durations help reduce thermal injury to the epidermis. When a 100-millisecond cryogen spurt is applied, thermal injury to the epidermis can be prevented in ex-vivo skin types I-IV when irradiated at higher incident dosages (15-20 J/cm(2)) than those currently used in clinical settings. Further studies on optimizing the CSC parameters in conjunction with the laser irradiation parameters are needed to protect skin types V-VI from thermal injury to the epidermis.

Methodology for characterizing heat removal mechanism in human skin during cryogen spray cooling.

Cryogen spray cooling (CSC) reduces epidermal damage during laser treatment of various dermatoses. The goal of this study was to determine the heat removal mechanism in skin and quantify the amount in response to CSC. Thermocouples were imbedded in four model substrates with a range of thermal diffusivities, greater than three orders of magnitude in difference, to measure the temperature profiles in response to CSC and sapphire contact cooling, which removes heat completely by conduction. An algorithm solving an inverse heat conduction problem was subsequently used to quantify the amount of heat removal from the substrates using the measured temperatures. The interface thermal conductance and internal temperatures within the substrates were computed by a finite difference algorithm that solved the heat conduction equation. Results verify a marked increase in heat removal and interface thermal conductance with increasing thermal diffusivity. By estimation from the model substrate results, heat removal and interface thermal conductance values for skin were obtained. Data demonstrate that during CSC, evaporation is the dominant heat transfer mechanism in materials with higher thermal diffusivities; however, conductive cooling dominates in substrates with lower thermal diffusivities such as skin.