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.

The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [³H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.

Background: One of the most sensitive methods for DNA double-strand break (DSB) detection is the assessment of phosphorylated histone protein 2AX (γH2AX), which can be visualized as γH2AX focus surrounding the damage site. Quantification of γH2AX offers a variety of potential diagnostic applications being on the brink of introduction into clinical routine. Therefore, further assay optimization and automation is required to enable standardized, high-throughput γH2AX analysis. To quantify γH2AX levels, different immunoassay techniques can be performed, each showing specific characteristics regarding assay sensitivity and sample processing. Although microscopic foci quantification is considered the most sensitive approach, data of direct comparison of multiple γH2AX immunoassay techniques are rare. In the current study we compared γH2AX quantification by different methods, including automated fluorescence microscopy, flow cytometry as well as immunoblotting. Further, we discussed assay-specific advantages and disadvantages. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were exposed to various concentrations of the DNA DSB-inducing, cytostatic drug etoposide for one hour. Subsequently, γH2AX levels were assessed by flow cytometry, immunoblotting and automated microscopy. Results: Automated fluorescence microscopic foci quantification revealed the lowest limit of detection (LoD) (0.53 µM etoposide). More than 10-fold higher etoposide concentrations were required to distinguish γH2AX values form background signal by fluorescence intensity-based methods like flow cytometry. Immunoblotting showed the poorest LoD of all three techniques. Conclusions: In contrast to flow cytometry and immunoblotting, automated fluorescence γH2AX foci quantification showed the lowest LoD. This low LoD allows the assessment and follow-up of patients with respective antitumor therapy. Thus, to apply the most suitable γH2AX analysis, specific assay characteristics must be considered.

Janus kinase inhibitors (JAKis) represent a new strategy in rheumatoid arthritis (RA) therapy. Still, data directly comparing different JAKis are rare. In the present in vitro study, we investigated the immunomodulatory potential of four JAKis (tofacitinib, baricitinib, upadacitinib, and filgotinib) currently approved for RA treatment by the European Medicines Agency. Increasing concentrations of JAKi or methotrexate, conventionally used in RA therapy, were either added to freshly mitogen-stimulated or preactivated peripheral blood mononuclear cells (PBMC), isolated from healthy volunteers. A comparable, dose-dependent inhibition of lymphocyte proliferation was observed in samples treated with tofacitinib, baricitinib, and upadacitinib, while dosage of filgotinib had to be two orders of magnitude higher. In contrast, antiproliferative effects were strongly attenuated when JAKi were added to preactivated PBMCs. High dosage of upadacitinib and filgotinib also affected cell viability. Further, analyses of DNA double-strand break markers γH2AX and 53BP1 indicated an enhanced level of DNA damage in cells incubated with high concentrations of filgotinib and a dose-dependent reduction in clearance of radiation-induced γH2AX foci in the presence of tofacitinib or baricitinib. Thereby, our study demonstrated a broad comparability of immunomodulatory effects induced by different JAKi and provided first indications, that (pan)JAKi may impair DNA damage repair in irradiated PBMCs.

Automated γH2AX foci quantification did not show evidence of increased DNA double-strand breaks after unenhanced or contrast-enhanced in vivo MR imaging at any investigated MR condition, including MR systems with different static magnetic field strengths (1 T, 1.5 T, 3 T, 7 T). PurposeTo determine the impact of different magnetic field strengths (1, 1.5, 3, and 7 T) and the effect of contrast agent on DNA double-strand–break (DSB) formation in patients undergoing magnetic resonance (MR) imaging.Materials and MethodsThis in vivo study was approved by the local ethics committee, and written informed consent was obtained from each patient. To analyze the level of DNA DSBs, peripheral blood mononuclear cells were isolated from blood samples drawn directly before, as well as 5 minutes and 30 minutes after MR imaging examination. After performing γH2AX immunofluorescence staining, DSBs were quantified with automated digital microscopy. MR group consisted of 43 patients (22 women, 21 men; mean age, 46.1 years; range, 20–77 years) and was further subdivided according to the applied field strength and administration of contrast agent. Additionally, 10 patients undergoing either unenhanced or contrast material–enhanced computed tomography (CT) served as positive control subjects. Statisti...

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double‐strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug‐induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter‐individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P‐gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB‐inducing agent etoposide (ETP) are known P‐gp substrates. In this respect, modulation of MDR by P‐gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP‐induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB‐positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB‐inducing cytostatics. © 2015 International Society for Advancement of Cytometry