Douglas L. Heintzelman

Near-Infrared Raman Spectroscopy for in Vivo Detection of Cervical Precancers

This study evaluates the potential of near-infrared Raman spectroscopy for in vivo detection of squamous dysplasia, a precursor to cervical cancer. A pilot clinical trial was carried out at three clinical sites. Raman spectra were measured from one colposcopically normal and one abnormal area of the cervix. These sites were then biopsied and submitted for routine histologic analysis. Twentyfour evaluable measurements were made in vivo in 13 patients. Cervical tissue Raman spectra contain peaks in the vicinity of 1070, 1180, 1195, 1210, 1245, 1330, 1400, 1454, 1505, 1555, 1656, and 1760 cm−1. The ratio of intensities at 1454 to 1656 cm−1 is greater for squamous dysplasia than all other tissue types, while the ratio of intensities at 1330 to 1454 cm−1 is lower for samples with squamous dysplasia than all other tissue types. A simple algorithm based on these two intensity ratios separates high-grade squamous dysplasia from all others, misclassifying only one sample. Spectra measured in vivo resemble those measured in vitro. Cervical epithelial cells may contribute to tissue spectra at 1330 cm−1, a region associated with DNA. In contrast, epithelial cells probably do not contribute to tissue spectra at 1454 cm−1, a region associated with collagen and phospholipids.

Optimal Excitation Wavelengths for In Vivo Detection of Oral Neoplasia Using Fluorescence Spectroscopy

Abstract There is no satisfactory mechanism to detect premalignant lesions in the upper aero-digestive tract. Fluorescence spectroscopy has potential to bridge the gap between clinical examination and invasive biopsy; however, optimal excitation wavelengths have not yet been determined. The goals of this study were to determine optimal excitation–emission wavelength combinations to discriminate normal and precancerous/cancerous tissue, and estimate the performance of algorithms based on fluorescence. Fluorescence excitation–emission matrices (EEM) were measured in vivo from 62 sites in nine normal volunteers and 11 patients with a known or suspected premalignant or malignant oral cavity lesion. Using these data as a training set, algorithms were developed based on combinations of emission spectra at various excitation wavelengths to determine which excitation wavelengths contained the most diagnostic information. A second validation set of fluorescence EEM was measured in vivo from 281 sites in 56 normal volunteers and three patients with a known or suspected premalignant or malignant oral cavity lesion. Algorithms developed in the training set were applied without change to data from the validation set to obtain an unbiased estimate of algorithm performance. Optimal excitation wavelengths for detection of oral neoplasia were 350, 380 and 400 nm. Using only a single emission wavelength of 472 nm, and 350 and 400 nm excitation, algorithm performance in the training set was 90% sensitivity and 88% specificity and in the validation set was 100% sensitivity, 98% specificity. These results suggest that fluorescence spectroscopy can provide a simple, objective tool to improve in vivo identification of oral cavity neoplasia.

Characterization of the Autofluorescence of Polymorphonuclear Leukocytes, Mononuclear Leukocytes and Cervical Epithelial Cancer Cells for Improved Spectroscopic Discrimination of Inflammation from Dysplasia

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho‐ and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250–550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl‐Met‐Leu Phe (1 μM) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: Results from an animal model

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

Optimal visual perception and detectionof oral cavity neoplasia

The most common way to detect disease is by visual inspection of the suspect tissue. However, the human eye is not optimized for this task because the perceived spectrum of light is divided into three channels, all of which have overlapping spectral sensitivity curves. Here, we present new methods to optimize visually perceived contrast based on spectral differences between normal and abnormal tissue. We apply these methods to the perception of fluorescence emission from the oral cavity. Abnormalities in the oral cavity are optimally perceived when the excitation is between 420-440 nm. To optimally visualize fluorescence at 340-nm excitation, the emission should be observed through a blue bandpass filter transmitting light at 430 nm.

Alternative chromophores for use in light-activated surgical adhesives: optimization of parameters for tensile strength and thermal damage profile

The use of indocyanine green-doped albumin protein solders has been shown to vastly improve the anastomotic strength that can be achieved by laser tissue repair techniques, while at the same time minimizing collateral thermal tissue damage. However, the safety of the degradation products of the chromophore following laser irradiation is uncertain. Therefore, we studied the feasibility of using alternative chromophores in terms of temperature rise at the solder/tissue interface, the extent of thermal damage in the sourrounding tissue, and the tensile strength of repairs. Biodegradable polymer scaffolds of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid), using a solvent-casting and particulate-leaching technique. The porous scaffold acted as a carrier to the traditional protein solder composition of serum albumin and an absorbing chromophore mixed in deionized water. Two commonly used chromophores, indocyanine green and methylene blue were investigated, as well as blue and green food colorings. Temperature rise at the solder surface and at the interface between the solder and tissue were monitored by an IR temperature monitoring system and a type-K thermocouple, respectively, and the extent of thermal damage in the underlying tissue was determined using light microscopy. As expected, temperature rise at the solder/tissue interface, and consequently the degree of collateral thermal tissue damage, was directly related to the penetration depth of the laser light in the protein solder. Optimal tensile strength of repairs was achieved by selecting a chromophore concentration that resulted in a temperature of 66 ± 3°C at the solder/tissue interface.

Comparison of scaffold-enhanced albumin and n-butyl-cyanoacrylate adhesives for joining of tissue in a porcine model

An ex vivo study was conducted to compare the tensile strength of tissue samples repaired using three different techniques: (i) application of a scaffold-enhanced light-activated albumin protein solder, (ii) application of a scaffold-enhanced n-butyl-cyanoacrylate adhesive, and (iii) repair via conventional suture technique. Biodegradable polymer scaffolds of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) and salt particles using a solvent-casting and particulate-leaching technique. Group I porous scaffolds were doped with protein solder composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. Group II scaffolds were doped with n-butyl-cyanoacrylate, and required no light-activation. No stay sutures were required for Group I or II experiments. Group III repairs were performed using a single 4-0 suture. Thirteen organs were tested ranging from skin to liver to the small intestine, as well as the coronary, pulmonary, carotid, femoral and splenic arteries. Acute breaking strengths were measured and the data were analyzed by Student’s T-test. Using the protein solder of Group I, repairs formed on the ureter were most successful followed by small intestine, sciatic nerve, spleen, atrium, kidney, muscle, skin and ventricle. The strongest vascular repairs were achieved in the carotid artery and femoral artery. Overall, the tensile strength of Group III repairs performed via suture techniques were equivalent in magnitude to that of Group I repairs, however, a larger variance was observed in the suture repair group. Group II repairs utilizing the cyanoacrylate-doped scaffold all performed extremely well. Bonds formed using the Group II adhesive were approximately 30% stronger than Group I and III organ repairs and approximately 20% stronger than Group I and III vascular repairs. Application of the polymer scaffold assists in tissue alignment and reduces problems associated with adhesive runaway from the repair site. Scaffold-enhanced adhesives could possibly be used as a simple and effective method to join tissue together quickly and effectively in an emergency situation, or as a substitute to mechanical sutures or staples in many clinical applications.

Alternative chromophores for use in light-activated surgical adhesives

A study was conducted to determine the feasibility of using alternative chromophores in light-activated surgical adhesives. Two commonly used chromophores, indocyanine green (ICG), and methylene blue (MB) were investigated, as well as three different food colorings: red #40, blue #1, and green food coloring consisting of yellow #5 and blue #1. The study consisted of three components. First, the absorption profiles of the five chromophores, both diluted in deionized water and bound to protein, were recorded with a UV-Vis-NIR spectrophotometer. Second, the effect of accumulated thermal dosages on the stability of the absorption profiles was investigated. Third, the stability of the absorption profiles of the chromophore solutions when exposed to ambient light for an extended period of time was investigated. The peak absorption wavelengths of ICG, MB, red #40, and blue #1, were found to be 780 nm, 665 nm, 500 nm, and 630 nm respectively. The green food coloring had two absorption peaks at 417 nm and 630 nm, corresponding to the two dye components comprising this color. The peak absorption wavelength of the ICG shifted to 805 nm when bound to protein. ICG and MB showed a significant decrease in absorbance units with increased time and temperature when heated to temperatures up to 100 degrees C. Negligible change in absorption with accumulated thermal dose was observed for any of the three food colorings investigated. Photobleaching was observed in both ICG and MB solutions with exposure to a white light source. An 88% decrease in absorption was seen in ICG deionized water solution after 7 days of exposure with a corresponding 33% decrease in absorption seen in the MB deionized water solution. A negligible drop in absorption was observed from exposure to ambient light for a 12-week period with the three food colorings investigated.

Cancer screening through the use of enhanced visual systems

The human eyes are not made to detect disease, however visual perception is the most common screening method for early cancer detection. With optimal illumination and observation configuration there is significant improvement of optical contrast between normal and pre-cancerous tissue in the oral cavity, both for reflected and fluorescent light.

Relationship between tissue redox potentials and hemoglobin oxygenation based on fluorescence of NADH/FAD and absorption spectroscopy of oxy/deoxy hemoglobin

Neoplastic changes in tissues are reflected by biochemical (tissue metabolism, blood oxygenation, blood volume) and morphological changes. These changes can be monitored non invasively using fluorescence / reflectance spectroscopy in the ultraviolet / visible spectral regions.