Shigehito Hayashi

Development of fluorescence-emitting antibody labeling substance by near-infrared ray excitation

Abstract Indocyanine green succinimidyl esters, near-infrared labeling dyes, were synthesized. These reagents were indocyanine green derivatives possesing N-hydroxysuccinimidyl groups capable of reacting with proteins. The absorption maximum of indocyanine green-labeled human IgG was 785 nm, and its fluorescent excitation and emission maxima were 768 nm and 807 nm, respectively.

Antibodies labeled with fluorescence-agent excitable by infrared rays

Abstract: Endoscopy is not significantly better than fiberscopy for the diagnosis of minute cancers of the digestive tract. However, labeling of these lesions with an agent that can be detected videoendoscopically, with subsequent computer processing of the electronic signals, should facilitate endoscopic diagnosis of microlesions. We developed an antibody labeled with an indocyanine green(ICG) derivative that has a specific fluorescence emission at 807 nm upon excitation at 768 nm. The physiochemical characteristics of this labeled antibody resemble those of ICG. The activity of the antibody is suitable for immunohistochemical staining, and the antibody fluoresces under infrared ray excitation. This antibody should prove useful for performing vital immunostaining for infrared endoscopy.

Reflected illumination-type imaging system for the development of infrared fluorescence endoscopy

Fluorescent labeled monoclonal antibodies against carcinoembryonic antigen (CEA) may provide a specific label for lesions of the GI tract. We previously developed an indocyanine green (ICG) derivative (ICG N‐hydroxy sulfo succinimide ester; ICG‐sulfo‐Osu) as a fluorescent label that is excited by infrared rays and is suitable for vital immunohistochemical staining. We also previously developed a transmitted illumination‐type (transmitted type) fluorescence imaging system that uses infrared rays to detect ICG‐sulfo‐Osu. In this paper we describe a new reflected illumination‐type (reflected type) imaging system for infrared fluorescence endoscopy. We tested the efficacy of this system on sections of human esophagus and normal skeletal muscle stained with ICG‐sulfo‐Osu labeled anti‐epithelial membrane antigen (EMA) antibody, and on sections of human gastric cancer tissue stained with ICG‐sulfo‐Osu labeled anti‐CEA antibody. Infrared fluorescent images were obtained with both fluorescent antibodies, and results correlated well with oxidized DAB‐positive sites. Vital immunohistochemical staining of micro cancers should be detectable by exciting an ICG‐sulfo‐Osu labeled antibody specific to the tumor cells with infrared rays, using this reflected type imaging system.

Development of agents for reinforcement of fluorescence on near-infrared ray excitation for immunohistological staining

Fluorescence intensity of indocyanine green (ICG) derivative (ICG-sulfo-OSu) was too low for its use to detect microlesions. Therefore, we examined the effects of reinforcement agents on ICG-sulfo-OSu labeled antibodies. Solutions of distearoylphosphatic acid sodium salt (DSPA) and octylglucoside (OG) in physiological phosphate buffered saline (PBS) were found to increase the intensity of fluorescence of ICG-sulfo-OSu labeled antibodies, with shift in the fluorescence peak wavelength from 804 to 821 nm.

Development of an Imaging System Using Fluorescent Labeling Substances Excited by Infrared Rays

Abstract: An indocyanine green (ICG) derivative (ICG N‐hydroxy sulfosuc‐cinimide ester; ICG‐sulfo‐OSu) has been developed as an antibody labeling substance suitable for vital immunohistochemical staining. However, an appropriate fluorescence imaging system for ICG‐sulfo‐OSu using infrared rays has not as yet been reported. Therefore, we developed such a system. The absorption maxima of ICG and ICG‐sulfo‐OSu in buffer solution are both at 795 nm, the excitation maxima of their fluorescence spectra in buffer solution at 768 nm, and their emission maxima at 807 nm. An imaging system using an excitation filter with transmission at 710‐790 nm and a barrier filter with transmission at 810‐920 nm was constructed, and fluorescent images of ICG‐sulfo‐OSu labeled anti‐epithelial membrane antigen (EMA) antibody were obtained with this system. Thus, vital immunohistochemical staining of microcancers under infrared ray excitation should now be possible, by exciting an ICG‐sulfo‐OSu labeled antibody specific to the tumor cells with infrared rays, using this imaging system.

Vital immunohistochemical staining for a novel method of diagnosing micro-cancer. Examination of immunohistochemical staining of non-fixed fresh tissue.

It becomes possible to establish a novel diagnostic method for micro-cancer by modulating the signals from the lesion, if lesions can be labeled with substances which can be detected by video endoscopy. The authors have already succeeded in synthesizing indocyanine green (ICG) derivatives for a fluorescent labeling substance which emits near-infrared rays. Before the antibodies labeled by these substances can be used, it is necessary to establish a method of vital immunohistochemical staining. So, we investigated the responses of antibodies exposed to non-fixed fresh tissue specimens as a basic study on vital immunohistochemical staining. The responses of fresh esophageal and gastric specimens (biopsied or surgically resected) to immunohistochemical staining with anti-epithelial membrane antigen (EMA) antibodies under various conditions using the ABC method were examined. These tissue specimens were stained immunohistochemically, and incubated with anti-EMA antibodies for 10 and 30 minutes (esophagus), and for 60 and 120 minutes (stomach) at 37 degrees C. These results suggest that vital immunohistochemical staining is possible under optimum conditions. If an infrared fluorescent endoscopy catching this excited fluorescence can be developed, it will be possible to establish a new endoscopic diagnostic method on the basis of vital immunohistochemical staining.

Serum amino‐terminal propeptide of type III procollagen and 7S domain of type IV collagen correlate with hepatic iron concentration in patients with chronic hepatitis C following α‐interferon therapy

Background: It has been reported that chronic infection with hepatitis C virus is associated with excess iron deposits in the liver of subjects who are neither alcoholics nor recipients of blood transfusions. However, little is known about the relationship between hepatic iron concentration (HIC) and the serum levels of hepatic fibrogenesis markers, which were caused by interferon therapy for chronic hepatitis C. Therefore, changes in the serum amino‐terminal propeptide of type III procollagen (P‐III‐P) and the 7S domain of type IV collagen (7S‐IV) in 16 patients treated with α‐interferon (IFN‐α) were studied, and their HIC and histological assessment evaluated. Hepatic iron concentrations were measured by using liver biopsy specimens obtained before and 6 months after the cessation of treatment.

Usefulness and Limitations of Endoscopic Ultrasonography in Diagnosis of Adenomyomatosis of the Gallbladder

Abstract: Although endoscopic ultrasonography (EUS) is commonly used to examine lesions in the gallbladder, its ability to accurately diagnose adenomyomatosis (ADM) has not been evaluated. We compared the accuracy of EUS and various other imaging techniques in the diagnosis of ADM. Thirty‐one patients undergoing cholecystectomy after elevated lesions were found in the gallbladder by various imaging techniques were studied retrospectively. Based on histopathologic examination, the sensitivity and specificity of EUS were evaluated in ADM. The sensitivity of abdominal ultrasonography, CT and ERCP in ADM were also evaluated. Of the 11 patients diagnosed with ADM by EUS, 9 cases were confirmed histologically (81.8%). Of the 11 patients with histologic diagnosis of ADM, 9 were accurately diagnosed preoperatively by EUS (81.8%). EUS proved more reliable for diagnosis of ADM than the other imaging techniques. Important diagnostic features include preservation of the three‐layered structure of the gallbladder, wall‐thickening exceeding 3 mm, and 2 or more Rokitansky‐Aschoff sinuses occurring within a 1 cm area of the gallbladder.