Novel image-based titration method for potency testing of diphtheria toxoid in Vero cell assay.

Protection of workers and consumers demands that safety testing accurately detects chemicals and products that can cause injury or disease. Chemicals make our lives more comfortable, but accidental and improper exposures to chemical substances continue to have a significant public health impact. According to the Institute of Medicine (2004), more than 4 million poisonings occur annually in the United States, and poisonings are the second leading cause of injury-related deaths, exceeded only by automobile accidents. Common household products such as household cleaners cause about 125,000 eye injuries each year. Skin diseases, injuries, and disorders, including contact dermatitis and allergic contact dermatitis from chemicals, are the most common category of occupational illness. Sixteen years ago the National Institutes of Health (NIH)Revitalization Act (1993) was signed into law with a little-noticed directive to the National Institute of Environmental Health Sciences (NIEHS) to develop a process to achieve regulatory acceptance of scientifically valid alternatives to animal-based safety testing. That directive gave rise to an interagency effort that has become increasingly effective in identifying, evaluating, and validating new test methods, including alternative methods, that reduce, refine, and replace animal testing—commonly referred to as “the 3Rs.” The NIEHS, along with 14 other U.S. Federal agencies, collectively known as the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM 2009b; ICCVAM Authorization Act of 2000), has made important and substantial progress benefiting both public health and animal welfare. ICCVAM’s focused efforts have resulted in approved alternative test methods for many types of product safety testing, including the four most commonly conducted safety tests: acute oral toxicity, dermal irritation/corrosion, ocular irritation/corrosion, and allergic contact dermatitis. The first test method evaluated and recommended by ICCVAM was a mechanism-based assay for allergic contact dermatitis testing that uses fewer animals and eliminates pain and distress compared to the traditional assay. The comprehensive ICCVAM evaluation served a key role in achieving rapid international acceptance and widespread use of this alternative method. Since that time, ICCVAM has contributed to the national and/or international regulatory acceptance of 27 alternative safety testing methods, including 17 that do not use live animals (ICCVAM 2009b). In April 2009, an ICCVAM initiative fostered an international agreement between the United States, Canada, Japan, and the European Union that is expected to further reduce animal use in product toxicity testing worldwide (Hood 2009; ICCVAM 2009a; NIEHS 2009). The agreement involves globally coordinated, high quality validation studies and peer reviews executed using a transparent process that should speed the international adoption of alternative toxicity testing methods. In the United States, federal law requires that new test methods must be determined to be valid for their proposed regulatory use before their adoption by regulatory agencies (ICCVAM Authorization Act of 2000). The law also stipulates that the new alternative test methods must provide equivalent or improved protection compared to existing methods. ICCVAM’s comprehensive scientific evaluations address these legal requirements, expediting acceptance of new test methods, and fulfilling a vital role in assisting agencies in meeting regulatory acceptance requirements. Promoting the use of accepted alternative methods has—and will continue to have—a huge positive impact on animal welfare. Thanks to the work done by ICCVAM and other organizations, animals are no longer required for many testing scenarios and the number of animals required for safety tests has been dramatically reduced. Where animals must still be used, many test methods have now been refined to significantly reduce or avoid most pain and distress. Most importantly, ICCVAM’s careful evaluation of the usefulness and limitations of these alternative methods will ensure that their proper use will in fact continue to support equal or better protection of people, animals, and the environment. Some 50 years ago in a book titled The Principles of Humane Experimental Technique, two British scholars named William Russell and Rex Burch described the concept of the 3Rs as a way to advance animal welfare (Russell and Birch 1959). They would undoubtedly be pleased to learn of the progress forged by ICCVAM.

ICCVAM: Birnbaum and Stokes Respond

Many types of medical and scientific experiments acquire raw data in the form of images. Various forms of image processing and image analysis are used to transform the raw image data into quantitative measures that are the basis of subsequent statistical analysis. In this article we describe the SimpleITK R package. SimpleITK is a simplified interface to the insight segmentation and registration toolkit (ITK). ITK is an open source C++ toolkit that has been actively developed over the past 18 years and is widely used by the medical image analysis community. SimpleITK provides packages for many interpreter environments, including R. Currently, it includes several hundred classes for image analysis including a wide range of image input and output, filtering operations, and higher level components for segmentation and registration. Using SimpleITK, development of complex combinations of image and statistical analysis procedures is feasible. This article includes several examples of computational image analysis tasks implemented using SimpleITK, including spherical marker localization, multi-modal image registration, segmentation evaluation, and cell image analysis.

This study was aimed at determining whether tumour DNA content measured by cell image analysis could provide additional prognostic information when compared to that provided by flow cytometry. Sections cut from paraffin blocks of tumours from 101 patients with node negative breast cancer were analysed by both methods and the results related to other prognostic variables and to patient relapse and overall survival. DNA ploidy measured by flow cytometry classified 46 tumours as diploid and 55 as aneuploid, whereas by cell image analysis 30 were diploid and 71 aneuploid (P less than 0.002). There were 20 tumours with discrepancies between the two methods; 18 of these were tumours with only one peak in flow analysis, but determined to be aneuploid with image analysis. DNA content as measured by both methods was significant for predicting relapse and survival by log-rank test, as were tumour histological grade, c-erbB-2 expression and tumour size. Multivariate analysis showed DNA ploidy measured by flow cytometry to be the only variable of independent significance (P less than 0.02) for both relapse and overall survival. Compared with cell image analysis, flow cytometry demonstrated a significantly higher proportion of diploid tumours, which may be related to differences in the internal standards applied to each method. We suggest that cell image analysis techniques can provide more sensitive information on the DNA content of tumour cells by direct measurement of nuclear DNA density of both normal lymphocytes and tumour cells in the same section. However, although image analysis appears to be more sensitive than flow cytometry in detecting DNA aneuploidy, the image technique appears to lack the specificity of flow cytometry in correlation with clinical outcome.

In 1997, the National Institute of Environmental Health Sciences (NIEHS) established the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), an ad hoc federal interagency committee to address the growing need for obtaining regulatory acceptance of new toxicological test methods. The thought was that simultaneous agency evaluation of new methods that addressed the 3Rs (reduction, refinement, and replacement) of animal testing by an interagency group could greatly speed up and harmonize the cross-agency acceptance and adoption of new methods into federal toxicity testing guidelines. This activity was codified into law in 2000 by passage of the ICCVAM Authorization Act (2000). The Act specified 15 agencies (such as the Food and Drug Administration, U.S. Environmental Protection Agency, Consumer Product Safety Commission, Department of Transportation, Occupational Safety and Health Administration, and U.S. Department of Agriculture) that would constitute ICCVAM. The Act also prescribed specific duties intended to facilitate review and acceptance of test methods, established an external scientific advisory committee, and required the director of the NIEHS to establish ICCVAM under the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), which currently exists as a functional unit within the Division of the NTP at the NIEHS. Over the past 15 years, ICCVAM has successfully evaluated and recommended numerous alternative test methods for regulatory use (NTP 2012). However, the lack of implementation of ICCVAM-recommended methods has been an area of increasing concern. The NIEHS has worked proactively with our ICCVAM partners to identify promising methods, encouraged and aided test developers in building a case for validating their methods, sometimes provided financial support through competitive Small Business Innovation Grants, and held workshops and engaged our federal and international partners to promote acceptance and use of test methods in specific areas of toxicology (e.g., ocular toxicity and skin sensitization). Even so, regulatory use of alternative methods has still lagged behind. Critics have repeatedly pointed out that alternative test methods have not been accepted for regulatory decision making and that the expectations for real reductions in animal use in toxicology testing have always outpaced the documented progress. It has become clear that it is time to change our approach. The NIEHS is beginning to move forward with a different philosophy toward ICCVAM. Rather than the NIEHS directing the activities of ICCVAM through NICEATM, the interagency agenda will now be driven by the partner regulatory agencies—the agencies that will ultimately implement the ICCVAM-recommended methods. Regulatory agencies are required by statute to use toxicology test information for a variety of purposes, including labeling and registration, and these requirements are not uniform. The ICCVAM Authorization Act acknowledges that some alternative test methods promoted by ICCVAM, while deemed valid, may not meet specific needs of a regulatory agency. With ICCVAM regulatory agencies taking ownership of the process, there should be a better match between the alternative test methods validated and the tests required to meet regulatory guidelines. Toxicology testing is shifting from a primary focus on adverse phenotypic observations in animals to mechanism-based biological outcomes in vitro, and the NIEHS is embracing this paradigm shift through its participation in the multiagency Tox21 consortium (Collins et al. 2008). NICEATM will expand its scope and concentrate its resources on providing bioinformatic and computational toxicology support to NIEHS Tox21 projects. With its purpose of transforming toxicology by shifting from in vivo animal studies to in vitro assays, in vivo assays in lower organisms, and computational modeling for toxicity assessments, Tox21 has the real potential to result in dramatic changes in the numbers and types of organisms used for toxicology testing. A stronger interface of NICEATM with Tox21 will better position ICCVAM for addressing how data from these new methods can be integrated into the existing regulatory framework. We express our deep appreciation to William S. Stokes, who has served as the director of NICEATM since its inception. In December 2012, he retired from the Public Health Service after 33 years of dedicated federal service. His vision, persistence, and direction have been key to bringing NICEATM, ICCVAM, and the International Cooperation on Alternative Test Methods (ICATM) to their current stage of maturity. We are pleased that Warren Casey, who has served as deputy director of NICEATM, will now serve as the acting director. He is uniquely qualified for this role, having worked in the areas of toxicogenomics, mechanistic toxicology, and biomarker development in the pharmaceutical industry prior to joining the NIEHS. We look forward to this new approach to promoting the 3Rs—an approach that will be driven by regulatory agency needs while remaining responsive to the test method development community.

1.1 Automated microscopy systems Nowadays the combination of progressive information technologies, modern methods and algorithms of processing, analysis and synthesis of images together with medicine has resulted in the emergence of a new branch or field, called telemedicine. Telemedicine is distant diagnostics based on image analysis and processing of human organs cells. There are the following directions in this field: medical-biological research automation (that is computer systems design, which provides input, processing and analysis of images); development and application of algorithms of biomedical images pre-processing, which improves quality of images; image analysis (feature extraction, classifiers development, etc.) and data transfer network technologies. This problem has been studied by many scientists. Today the analysis of medical-biological specimens (cytological and histological smears) in diagnostic laboratories is conducted by visual inspection. This process is routine and labour intensive. That’s why the automated microscopy systems (AMSs) – software-hardware complexes appeared for the digital processing of microscopic images. AMS includes motorized controlled microscope, video camera, optical adapters, computer, and specialized software modules. In general the medical complexes for automated microscopy are made to provide higher productivity of medical work; improve analysis accuracy, availability of intensive labour-consuming and rare analyses, quality control, telemedicine; to collect and archive specimen image; and better learning, service and certification. AMS provides the following levels of microscopic analyses automations: 1. Visual analysis, documenting and telemedicine; 2. Analysis of images in order to determine the specimen characteristics; 3. Automation of movements and inspection of specimen. Morphometry of cells is only the one of the automated methods in AMS. Basic steps of cytological and histological analysis are the following: selection of an object of investigation, preparation for microscope inspection, microscopy techniques application, qualitative and quantitative analysis of images. Type, structure and functions of the system are dictated by actual task, objects class of research and financial ability of user. Problem definition. The actual tasks are projecting of AMS hardware and software structures based on the hardware components existing in the market and development of the own software for histological and cytological image analysis.

The aim of this study is to determine the thickness distribution of a food package using a non-destructive method. Initially, thickness measurements were carried out using an experimental procedure for thermoformed samples that were used for food packaging. Additionally, in this study, image analysis was used for the first time to determine the thickness distribution of the thermoformed products non-destructively. Image analysis software was employed for the estimation of thickness distribution. Measured thickness results were compared to those estimated using image analysis. Based on the results of the current study, image analysis may be an alternative method for non-destructive testing of thermoformed food packages even in a mass production line. Image analysis can be used to determine not only thickness distribution but also the weakest regions in a food package.

This study determined the validity of an image analysis program developed to score individual cells in human solid tumors labeled by proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdUrd). The program used nuclear size, grey level, and perimeter convexity to identify cells, and evaluated labeling by the fraction of nuclear area displaying positive immunostaining (MPB). Total cell number (TC) and BrdUrd or PCNA labeling index (LI) were evaluated in 142 images using visual (TCvisual, LIvisual) and image analysis (TC(IA), LI(IA)). Without the perimeter convexity criterion, image analysis resulted in a) TC(IA) equal to TCvisual in spite of the presence of various non-cellular objects and b) significant correlations between LI(IA) and LIvisual for PCNA and BrdUrd, although for these markers the LI(IA) were 4 and 6% lower than their respective LIvisual. Both visual and image analyses yielded significant inter-investigator variation among three investigators (coefficient of variation between 8.2 and 47.5%) and significant intra-investigator, inter-day variation (coefficients of variation between 3.8 and 51.8%). We conclude that image analysis using size, grey level and MPB is a valid alternative to visual scoring of PCNA and BrdUrd LI in individual cells.

BackgroundHigh content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study.ResultsThe cellular images of H4 neuroglioma cells exposed to different concentrations of CuO nanoparticles were sampled using IN Cell Analyzer 1000. A fully automated cellular image analysis system has been developed to perform the image analysis for cell viability. A multiple adaptive thresholding method was used to classify the pixels of the nuclei image into three classes: bright nuclei, dark nuclei, and background. During the development of our image analysis methodology, we have achieved the followings: (1) The Gaussian filtering with proper scale has been applied to the cellular images for generation of a local intensity maximum inside each nucleus; (2) a novel local intensity maxima detection method based on the gradient vector field has been established; and (3) a statistical model based splitting method was proposed to overcome the under segmentation problem. Computational results indicate that 95.9% nuclei can be detected and segmented correctly by the proposed image analysis system.ConclusionThe proposed automated image analysis system can effectively segment the images of human H4 neuroglioma cells exposed to CuO nanoparticles. The computational results confirmed our biological finding that human H4 neuroglioma cells had a dose-dependent toxic response to the insult of CuO nanoparticles.

DNA ploidy by image cytometry and karyotype in spontaneous abortion

Comparative mutagenecity tests with isoniazid in various mammalian test systems : Röhrborn, G., Institute of Anthropology and Human Genetics, University of Heidelberg, Heidelberg (Germany)

Animal testing for toxicity assessments has long been a point of contention due to concerns over animal welfare and ethics. As an integral component of product development and consumer safety, the evaluation of chemical toxicity is vital. Yet, the 21st century has witnessed a growing demand for alternative testing methods that can offer sustainable solutions without relying on animal experiments. This need has spurred major developed countries and international standardization organizations to pursue the development and standardization of novel testing approaches. Among these, organoid-based methods have emerged as a promising avenue for innovation.
 This study examines the concept of organoids, defining and categorizing alternative testing methods. It explores a broad spectrum of topics, including the standardization trends of alternative testing method regulations, organoid-based method standardization, and trends led by government entities, research institutions, academia, and corporations. It also examines the development of organ-specific manufacturing protocols for organoids. The research finds that international efforts to standardize alternative testing methods are in progress, with a particular focus on organoid-based strategies.
 The anticipated growth and evolution of such alternatives could revolutionize standard practices in safety assessments and drug efficacy evaluations. The standardization of organoid-based testing methods is poised to become a crucial milestone in advancing human health and environmental protection, driving the sector's growth and innovation. Moreover, advancements in organoid technology and the creation of models for various organs are likely to further refine alternative testing methods and introduce new evaluation techniques for drugs and chemicals. This study highlights to the biomedical community that standardizing new testing methods can address the ethical dilemmas of animal testing, underscoring a significant contribution towards ethical scientific practices.

Conventional cytology cannot discriminate between benign and malignant follicular neoplasms. Our study evaluated the diagnostic role of computer-assisted image analysis in the presurgical assessment of thyroid follicular neoplasms. Fifty-eight patients (14 males, 44 females, age range, 45-75 years) who underwent surgery for cytologic diagnosis of thyroid follicular neoplasm were studied. All patients were first evaluated on clinical grounds and assigned a high/low suspicion of malignancy on the basis of gender, age, and nodule size. Cell image analysis was subsequently performed using a Cytometrica BYK Gulden microscope image processor on Feulgen-stained thyroid cytologic smears. A different population of 50 benign and 50 malignant, histologically evaluated nodules was studied in order to establish image analysis criteria suggestive of thyroid malignancy. Ploidy histogram, proliferation index (PI), nuclear area coefficient of variation (NACV), and anisocariosis ratio (AR) were studied. Thyroid cancer was diagnosed in 16 of 58 follicular neoplasms. Only 7 of these lesions were clinically suspicious (43.7%), whereas 14 of 16 (87.5%) malignant tumors were identified by image analysis. Positive and negative predictive values of image analysis versus clinical evaluation were 46.6% versus 30.4% and 92.8% versus 74.3%, respectively. The distribution of ploidy pattern was different in benign versus malignant follicular neoplasms (chi2 8.25, p = 0.016), malignant lesions showing an increased frequency of heteroclonal aneuploid DNA content (37.5% vs. 7.1%). Increased PI (mean +/- standard deviation (SD) = 11.3 +/- 5.7 vs. 7.1 +/- 6.1; p < 0.01) and NACV (mean +/- SD = 25.28 +/- 1.89 vs. 20.14 0.91; p < 0.01) levels were also observed in malignant follicular neoplasms. In conclusion, computer-assisted image analysis may profitably support clinical evaluation in the assessment of thyroid follicular neoplasms.

This paper describes one model of the method of accelerated multi-factorial testing of roller bearing reliability, that represents the result of many years of work and research of the authors in the field of technical system effectiveness, and that treats, from the theoretical point of view, a completely new method of multi-factorial reliability testing, which the broader scientific and expert circles are not yet familiar with. The method of accelerated multi-factorial testing of roller bearing reliability, treated by this work, contains significant advantages with respect to all other methods of reliability testing, which have been used so far in the engineering reliability testing practice in the world..

This article briefly reviews three areas of research related to testing and the assessment of memory structures. The research is criticized from the stand point of either, (a) failing to provide achievement testing methods that yield useful diagnostic information for instruction or, (b) not being prac tical for instructional purposes. An alternative achievement testing method, based on research on reading comprehension, is proposed. Preliminary research on the alternative testing method is summarized.