Immunoassays are capable of measuring very small concentrations of substances in solutions and have an immense range of application. Enzyme-linked immunosorbent assay (ELISA) tests in particular can detect the presence of an infection, of drugs, or hormones (as in the home pregnancy test). Inference of an unknown concentration via ELISA usually involves a non-linear heteroscedastic regression and subsequent prediction, which can be carried out in a Bayesian framework. For such a Bayesian inference, we are developing informative prior distributions based on extensive historical ELISA tests as well as theoretical considerations. One consideration regards the quality of the immunoassay leading to two practical requirements for the applicability of the priors. Simulations show that the additional prior information can lead to inferences which are robust to reasonable perturbations of the model and changes in the design of the data. On real data, the applicability is demonstrated across different laboratories, for different analytes and laboratory equipment as well as for previous and current ELISAs with sigmoid regression function. Consistency checks on real data (similar to cross-validation) underpin the adequacy of the suggested priors. Altogether, the new priors may improve concentration estimation for ELISAs that fulfill certain design conditions, by extending the range of the analyses, decreasing the uncertainty, or giving more robust estimates. Future use of these priors is straightforward because explicit, closed-form expressions are provided. This work encourages development and application of informative, yet general, prior distributions for other types of immunoassays.

An in vivo near-infrared fluorescence (NIRF) imaging technique is described for therapy monitoring of ankle joints affected by collagen-induced arthritis, a model of human rheumatoid arthritis. Arthritis was induced in rats by intradermal injections of collagen and Freund's incomplete adjuvant. For in vivo imaging, the nonspecific NIR dye tetrasulfocyanine (TSC) was used. Prior to and after treatment with a nonsteroidal anti-inflammatory drug, meloxicam, or analgesic drug, tramadol hydrochloride (which served as no-therapy control), normalized fluorescence intensities of each ankle joint were measured. Additionally, each ankle joint was characterized by clinical arthritis scoring and histopathology. Over a 3-week treatment period, a significant difference in disease progression between animals treated with meloxicam and tramadol hydrochloride was detected. A statistically significant improvement in ankle joint pathology from high- or moderate-grade to moderate- or low-grade upon meloxicam therapy, as determined by clinical evaluation, translated into a significant decrease in fluorescence intensity. In contrast, all arthritic joints of the no-therapy control group deteriorated to high-grade arthritis with high-fluorescence intensities in NIRF imaging.

Optical imaging offers a high potential for noninvasive detection of cancer in humans. Recent advances in instrumentation for diffuse optical imaging have led to new capabilities for the detection of cancer in highly scattering tissue such as the female breast. We review recent developments in the detection of breast cancer in humans by fluorescent contrast agents. So far, the unspecific contrast agents indocyanine green (ICG) and omocyanine have been applied, whereas molecular probes for direct targeted imaging of this disease are still in preclinical research. We discuss recent improvements in the differentiation of malignant and benign lesions with ICG based on its enhanced extravasation in breast cancer. Whereas fluorescence imaging in thick tissue layers is hampered by strong light scattering, tissue surfaces can be investigated with high spatial resolution. As an example for superficial tumors, lesions of the gastrointestinal tract (GI) are discussed. In these investigations, protoporphyrin IX is used as a tumor-specific (due to its strong enhancement in tumor cells) target for spectroscopic identification and imaging. We present a time-gated method for fluorescence imaging and spectroscopy with strong suppression of tissue autofluorescence and show results on patients with Barrett's esophagus and with colitis ulcerosa.

The development of fluorescence applications in the life and material sciences has proceeded largely without sufficient concern for the measurement uncertainties related to the characterization of fluorescence instruments. In this first part of a two-part series on the state-of-the-art comparability of corrected emission spectra, four National Metrology Institutes active in high-precision steady-state fluorometry performed a first comparison of fluorescence measurement capabilities by evaluating physical transfer standard (PTS)-based and reference material (RM)-based calibration methods. To identify achievable comparability and sources of error in instrument calibration, the emission spectra of three test dyes in the wavelength region from 300 to 770 nm were corrected and compared using both calibration methods. The results, obtained for typical spectrofluorometric (0°/90° transmitting) and colorimetric (45°/0° front-face) measurement geometries, demonstrated a comparability of corrected emission spectra within a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based spectral correction when measurements and calibrations were performed under identical conditions. Moreover, the emission spectra of RMs F001 to F005, certified by BAM, Federal Institute for Materials Research and Testing, were confirmed. These RMs were subsequently used for the assessment of the comparability of RM-based corrected emission spectra of field laboratories using common commercial spectrofluorometers and routine measurement conditions in part 2 of this series (subsequent paper in this issue).

In the second part of this two-part series on the state-of-the-art comparability of corrected emission spectra, we have extended this assessment to the broader community of fluorescence spectroscopists by involving 12 field laboratories that were randomly selected on the basis of their fluorescence measuring equipment. These laboratories performed a reference material (RM)-based fluorometer calibration with commercially available spectral fluorescence standards following a standard operating procedure that involved routine measurement conditions and the data evaluation software LINKCORR developed and provided by the Federal Institute for Materials Research and Testing (BAM). This instrument-specific emission correction curve was subsequently used for the determination of the corrected emission spectra of three test dyes, X, QS, and Y, revealing an average accuracy of 6.8% for the corrected emission spectra. This compares well with the relative standard uncertainties of 4.2% for physical standard-based spectral corrections demonstrated in the first part of this study (previous paper in this issue) involving an international group of four expert laboratories. The excellent comparability of the measurements of the field laboratories also demonstrates the effectiveness of RM-based correction procedures.

Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p<0.005) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, INF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81%) than for MRI (100%/91%). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.

Due to its spectral and binding properties, the well-tolerated near-infrared absorbing and fluorescent dye indocyanine green (ICG) has found various applications in diffuse optical imaging of diseases in humans. Here, we review applications of this dye for the detection of breast cancer, for perfusion monitoring of the brain in ischaemic stroke and during cardiac surgery, and for the detection of rheumatoid arthritis in finger joints. We give a short overview of the properties of ICG which are relevant for diffuse optical imaging and summarise clinical applications of the dye. Then we describe the experimental methods used for contrast-enhanced imaging of breast cancer with ICG, and we discuss the results of studies on patients with breast lesions. Generally, after intravenous application an increased concentration of ICG in breast carcinomas was observed. In part, differences in ICG pharmacokinetics between malignant lesions, benign lesions and healthy tissue were reported. In a recent study, fluorescence measurements revealed significant differences in extra-vasation of ICG between malignant and benign breast lesions. In brain imaging, ICG has been used as a tracer to assess perfusion of the cortex. Measurements on stroke patients have been performed by comparing the arrival times of a bolus in the affected and in the healthy hemisphere of the brain. Furthermore, ICG boluses have been applied to monitor cerebral blood flow during cardiac surgery. We review the results of the available studies based on either continuous wave light sources, frequency domain techniques, or time domain techniques, and we discuss the depth selectivity of these methods. In one of the studies, fluorescence detection of ICG was investigated and compared to absorption detection used so far. When employed for the detection of rheumatoid arthritis in finger joints, the time course of the ICG distribution in the tissue after intravenous bolus injection was recorded by measuring ICG fluorescence in reflection geometry. We describe the features of the experimental technique and discuss the results of clinical studies performed so far. Finally, we summarise the properties of ICG exploited as contrast mechanisms in diffuse optical imaging.

AbstractThe sections in this article areIntroductionNon‐Invasive Fluorescence Imaging Techniques and Fluorescent Imaging AgentsExperimental Studies in Arthritis ModelsNonspecific Fluorescent Imaging Agents in Arthritis ModelsTargeted Fluorescent Imaging Agents in Arthritis ModelsActivatable Fluorescent Imaging Agents in Arthritis ModelsMonitoring Cell Migration in Arthritis ModelsClinical ApplicationsCritical Overview of Fluorescence Imaging in Arthritis

A new concept of tumor imaging is introduced using a siRNA-based probe that is capable of amplifying a specific endogenous fluorescence emission in cancerous tissue. In previous studies, we demonstrated a significant downregulation of Ferrochelatase (FECH) mRNA-expression in colorectal carcinomas leading to the accumulation of protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis. In this article, we report on first in vivo experiments in xenografted nude mice using folate-coupled liposomes or dendritic polyglycerolamine nanoparticles carrying ferrochelatase-siRNA to enhance PpIX-derived fluorescence in the tumor tissue. Tiny tumor foci could be monitored by the emission of PpIX fluorescence in vivo. Due to the omnipresence of the heme synthesis pathway, targeted application of ferrochelatase-siRNA may provide a general means for molecular imaging.From the Clinical Editor: A new concept of tumor imaging is presented in this paper using a siRNA-based probe detecting protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis previously demonstrated to accumulate in cancer tissue. (C) 2012 Elsevier Inc. All rights reserved.

We report on the development of a scanning non-contact brain imager, based on a novel technique in time-resolved nearinfrared spectroscopy, i.e. the null source-detector distance approach. Our concept is designed to image an area of about 10 cm2 with small adjustable scanning steps, i.e. a high density of mapping points can be realized. The feasibility of the proposed method was tested with a single-point confocal optical setup without beam scanning so far. A set of test measurements was performed on a liquid phantom with a small black polyvinyl chloride (PVC) cylinder as a target, which was translated in X direction to emulate the optical scanning and estimate lateral spatial resolution, and in Z direction to estimate the depth sensitivity of the instrument. The problem of dominance of early photons at null sourcedetector separation was solved by applying a fast time-gated detector to detect late only photons. Two fast-gated detectors, a newly developed state-of-art time-gated single-photon avalanche photodiode (tgSPAD) and commercially available fast-gated intensified CCD (iCCD) camera, were compared against each other. It was shown that, due to its better dynamic range, the tgSPAD is capable to detect later photons than the iCCD camera, and hence, a scanning system equipped with the time-gated SPAD has better depth sensitivity. Thus the time-gated SPAD is the detector of choice for further development of the non-contact confocal brain scanner.

BACKGROUND:Patients with long-standing ulcerative colitis require repeated endoscopies for early detection of neoplasias, which, however, are frequently missed by standard colonoscopy. Fluorescence-guided colonoscopy is known to improve the detection rate but the long-term effects of fluorescence-guided colonoscopy are unknown.METHODS:Colitis patients with negative findings at index fluorescence-guided colonoscopy entered a prospective long-term study with conventional colonoscopies at 2-year intervals. Risk and time to progression were evaluated. The positive predictive value was assessed in patients with neoplasias at index fluorescence-guided colonoscopy who underwent immediate total colectomy.RESULTS:Thirty-one patients with negative fluorescence-guided colonoscopy were surveyed for a mean of 7.8 ± 0.9 years. Neoplasia was observed in only two of them (6%) after 7 and 8 years of follow-up, respectively. Neoplasia at index fluorescence-guided colonoscopy was observed in 10 patients. In all of them, multiple flat low-grade intraepithelial neoplasia was diagnosed. At immediate colectomy performed in eight of them, the diagnosis of flat low-grade intraepithelial neoplasia was confirmed, corresponding to a positive predictive value of 100%. However, synchronous more advanced neoplasia was detected in three of the eight patients (38%). All patients, those with and those without neoplasia, were alive at the end of the study.CONCLUSIONS:Fluorescence-guided colonoscopy misses, in contrast to standard colonoscopy, few, if any, patients with neoplasia. Most neoplasia-negative patients remain negative during prolonged follow-up. However, when low-grade dysplasia is diagnosed by fluorescence-guided colonoscopy, colectomy is recommended because more than a third of the patients harbor synchronous, more advanced neoplasia.

Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ~70 sulfate groups) targets factors of inflammation (IC(50) of 3-6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by near-IR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1-3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.

BACKGROUND:Immunoassays are biochemical tests applied to measure even very small amounts of substance using the highly specific binding between an antibody and its antigen. They have a wide range of applications. The measurement however, might be associated with substantial uncertainty; this can have significant consequences for any diagnosis, or clinical decision. An international comparability study was thus performed to assess the sources of uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA.METHODS:In contrast to the original publication for this international comparability study, we reanalyse the data using Bayesian inference. This provides a statistically coherent approach to estimate ELISA concentrations and their associated uncertainties.RESULTS:The Bayesian uncertainties of individual ELISAs and laboratory estimates are considerably larger than previously reported uncertainties. The average concentrations estimated here differ from the ones estimated by each study participant. In general, this leads to different conclusions about the study. In particular, the inter- and intra-laboratory consistency is increased, and repeatability problems occur for fewer laboratories.CONCLUSIONS:Decisions which are based on plausible ranges of measurements (such as credible intervals), are generally superior to those solely based on point estimates (such as the mean). Reliable uncertainties are thus vital, and not only in metrology. In this paper, a general method is developed to derive concentration estimates and valid uncertainties for ELISAs. Guidance on applying this Bayesian method is provided and the importance of reliable uncertainties associated with ELISAs is underlined. The applicability and virtues of the presented method are demonstrated in the context of an international comparability study.

We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1(')-bis-(4-sulfobutyl) indotricarbocyanine-5,5(')-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.

The blood pool agent indo-cyanine green (ICG) has been investigated in a prospective clinical study for detection of rheumatoid arthritis using fluorescence imaging. Temporal behavior as well as spatial distribution of fluorescence intensity are suited to differentiate healthy and inflamed finger joints after i.v. injection of an ICG bolus.

The dye ICG was investigated in a clinical study for evaluation of rheumatoid arthritis using fluorescence imaging. Second moments of correlated time series of fluorescence intensities were analysed to differentiate healthy subjects from diseased.

RATIONALE AND OBJECTIVES:The aim of this study was to develop a new tool for the early detection of inflammatory joint diseases using fluorescence imaging in the near-infrared (NIR) spectral range following the intravenous administration of an unspecific contrast agent.MATERIALS AND METHODS:A laser-supported system for fluorescence imaging of finger joints was designed and constructed. Five patients and a corresponding number of volunteers were examined using 0.1 mg/kg by weight of indocyanine green as an unspecific contrast agent. Fluorescence images were acquired continuously over a period of 15 minutes. As a control, 1 day before optical imaging, all patients and volunteers underwent contrast-enhanced magnetic resonance imaging (MRI) at 0.2 T. On the basis of MRI findings, all examined joints were divided into four groups: no change and mild, moderate, and severe synovitis. The emitted fluorescence photons were quantified in different regions of interest covering the finger joints and finger tips. The normalized fluorescence intensity of contrast agents was compared with MRI findings as a proven standard.RESULTS:NIR dyes of the cyanine class are enriched in inflammatory joints and show a different kinetic behavior compared to normal joints after bolus injection. These findings demonstrate clearly the capability of contrast-enhanced fluorescence imaging to detect early changes caused by rheumatoid arthritis in finger joints. The NIR results were correlated with MRI findings (r = 0.84).CONCLUSION:Contrast-enhanced fluorescence imaging provides adequate information for the evaluation of inflammatory involvement of finger joints comparable to low-field MRI.

Background: Dysplasia in ulcerative colitis is frequently missed with 4-quadrant biopsies. An experimental setup recording delayed fluorescence spectra simultaneously With white light endoscopy was recently, developed.Objective: We compared detection of invisible flat intraepithelial neoplasia with protoporphyrin IX fluorescence and standard 4-quadrant biopsies.Design: Prospective, crossover design without randomization of the order of procedures.Setting: Gastroenterology Department, Humboldt University, Charite, Berlin, Germany.Patients: Forty-two patients with extensive ulcerative colitis of more than 10 years' duration were included.Interventions: Colonoscopy with 4-quadrant biopsies and targeted biopsies of macroscopic lesions and time-gated fluorescence-guided colonoscopy were performed 2 weeks apart by 2 blinded endoscopists. Three independent pathologists examined the biopsy specimens.Main Outcome Measurements: The primary Outcome criterion was detection rate of invisible flat intraepithelial neoplasia.Results: Invisible flat intraepithelial neoplasia was detected in 3 (7%) patients by white light 4-quadrant biopsies and in 10 (24%) patients by fluorescence-guided endoscopy (P = .02). The sensitivity and specificity for differentiating patients with and without dysplasia were 100% and 81%, respectively. Dysplastic and nondysplastic mucosa Could be discriminated with a sensitivity and specificity of 73% and 81%, respectively.Limitations: The trial was not randomized.Conclusion: The detection rate of intraepithelial neoplasia in patients with ulcerative colitis can be improved 1 by fluorescence-guided colonoscopy. (Gastrointest Endosc 2010;71:312-8.)