Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology.

Analysis of phosphorylated histone protein H2AX (cH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make cH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES V R platform to automatically analyze immunofluorescence images of cH2AX foci and compared the results with visual assessments. Dose-and time-dependent cH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on cH2AX foci formation in PBMCs. Apart from cH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping cH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of cH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 lM ETP demonstrated a peak in cH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of cH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for cH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage. V C 2013 International Society for Advancement of CytometryKey terms cH2AX foci; automated microscopy; image analysis; DNA double-strand breaks; etoposide; human PBMCs THE damage of DNA is a critical event able to affect cellular functions and development. Thus, it is essential for cells to maintain DNA integrity and repair such lesions effectively. Among different kinds of DNA lesion, double strand breaks (DSB) are considered to be the most critical type of DNA damage and misrepair can lead to tumorgenesis or cell death (1,2). To ensure detection and repair of DNA damage sites a variety of proteins are involved in different DNA damage response (DDR) pathways (3). After induction of DSB, the histone protein H2AX is rapidly phosphorylated at serine 139, termed cH2AX. Large amounts of cH2AX molecules form a focus in the

No abstract

No abstract

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.