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Willitzki A.,Otto Von Guericke University of Magdeburg | Hiemann R.,Lausitz University of Applied science | Peters V.,Medipan GmbH | Sack U.,University of Leipzig | And 8 more authors.
Clinical and Developmental Immunology | Year: 2012

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. © 2012 Annika Willitzki et al.


Farahati J.,Institute of Radiology | Roggenbuck D.,Medipan GmbH | Gilman E.,Institute of Radiology | Schtte M.,University of Duisburg - Essen | And 4 more authors.
Clinical Chemistry and Laboratory Medicine | Year: 2012

Background: The presence of thyroid peroxidase antibodies (TPOab) are reported to be associated with improved outcome among breast cancer patients. We evaluated the correlation between TPOab and diagnostic parameters among newly diagnosed breast cancer patients. Methods: Three hundred and fourteen newly diagnosed patients with breast cancer, diagnosed and treated in Bethesda Essen between January 2002 and June 2006, were included in this study; 258 (82.2) without TPOab (≤100 IU/mL) and 56 (17.8) with TPOab (>100 IU/mL). Blood analysis was performed to measure serum levels of carcinoembryonic antigen (CEA), cancer antigen 15-3 (CA-15-3), free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH) and TPOab by radioimmunoassay. Data regarding age, tumor size, grading, TNM classification, receptor status, lymph node, and distant metastases were collected and analyzed from patient reports. Statistics were performed using Pearson's χ2-test and logistic regression analysis. Results: There were no incidences of distant metastasis among 56 patients with TPOab, whereas 17 (6.6) of 258 cases without TPOab displayed distant metastases (p0.04). Logistic regression showed an inverse association of TPOab with CA-15-3 and CEA levels (p<0.001, respectively). Both groups, with and without TPOab, revealed no significant differences with respect to age, tumor size, grading, TNM classification, fT3, fT4, and receptor status. TPOab positive patients had higher TSH levels (2.55±3.58), compared to TPOab negative cases (1.20±1.15) (p<0.001). Conclusions: TPOab occurrence is associated with significantly lower frequency of distant metastases in breast cancer. TPOab level inversely correlates with the conventional tumor markers CA-15-3 and CEA. © 2012 by Walter de Gruyter Berlin Boston.


Roggenbuck D.,Medipan GmbH | Roggenbuck D.,Sudan University of Science and Technology | Somma V.,Medipan GmbH | Schierack P.,Sudan University of Science and Technology | And 4 more authors.
Lupus | Year: 2014

The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.


Werner L.,IBD Center | Werner L.,Tel Aviv Sourasky Medical Center | Sturm A.,Tel Aviv University | Roggenbuck D.,Krankenhaus Waldfriede | And 13 more authors.
Journal of Crohn's and Colitis | Year: 2013

Background and aims: The Crohn's disease (CD)-specific pancreatic auto-antibodies (PAB), have been recently identified to target glycoprotein 2 (GP2). Pouchitis is an inflammation of the small bowel developing in up to 60% of ulcerative colitis patients undergoing proctocolectomy and ileal pouch anal anastomosis. Occurrence of CD-specific antibodies was reported to be a predictor of pouchitis. We aimed to assess the prevalence of anti-GP2 antibodies (anti-GP2) in the serum and feces of pouch patients and to correlate them with clinical parameters. Furthermore, we examined mucosal expression of the GP2 protein in the pouch. Methods: Pouch patients were prospectively recruited and checked for clinical, endoscopic, and laboratory markers of inflammation. IgG and IgA anti-GP2 levels in serum and fecal samples were determined using ELISA. GP2 protein was assessed by immunohistochemistry. Results: Anti-GP2 was elevated in both serum and fecal samples of patients with inflamed compared to those with non-inflamed pouches and patients with familial-adenomatous polyposis after surgery (p < 0.05, respectively). Moreover, patients with CD-like complications exhibited significantly higher anti-GP2 titers than those without CD-like complications (p ≤ 0.01). High levels of anti-GP2 correlated with more frequent bowel movements per day and with the presence of at least one anti-glycan antibody (p ≤ 0.05). GP2 itself was more abundant in the mucosa of patients with chronic pouchitis. Conclusions: Anti-GP2 exists in the serum and feces of pouch patients and correlates with pouch inflammation, and presence of other serological markers. Thus, anti-GP2 may contribute to better stratification of pouchitis, more-so when the inflammation exhibits CD-like complications. © 2013 European Crohn's and Colitis Organisation.


PubMed | Charité - Medical University of Berlin and Medipan GmbH
Type: Journal Article | Journal: PloS one | Year: 2016

In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as -H2AX. Formation of -H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. -H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS).To evaluate the significance of -H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated -H2AX and 53BP1 foci detection.Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with -H2AX and 53BP1 specific antibodies. Nuclear -H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of -H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. -H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls.The median (range) number of -H2AX (0.04 [0-0.5]) and 53BP1 (0.005 [0-0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, -H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. -H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although -H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, -H2AX values of both groups overlapped and -H2AX did not correlate with the number or volume of CEL.-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.


PubMed | Otto Von Guericke University of Magdeburg, Sudan University of Science and Technology, University of Leipzig and Medipan GmbH
Type: Journal Article | Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology | Year: 2015

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 (H2AX). Furthermore, H2AX 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 H2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of H2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in H2AX 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 H2AX 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..


Runge R.,TU Dresden | Hiemann R.,Applied Information Sciences | Wendisch M.,TU Dresden | Kasten-Pisula U.,University of Hamburg | And 7 more authors.
International Journal of Radiation Biology | Year: 2012

Purpose: Assessment of phosphorylated histone H2AX (γH2AX) foci as a measure for double-strand breaks (DSB) is a common technique. Since visual interpretation is time-consuming and influenced by subjective factors, we adapted the pattern recognition algorithms of autoantibodies to automated reading of γH2AX foci. Materials and methods: DSB formation was assessed by detection of γH2AX foci after exposition of thyreocyte rat cell line to 188Re. We used pattern recognition algorithms of the automated fluorescence interpretation system AKLIDES® for evaluation of γH2AX foci. Manual investigation was performed by three laboratories involving five observers. The results were compared by determining correlation and inter-laboratory variability. Results: The study confirmed the adaptation of automated interpretation system AKLIDES® to automated assessment of γH2AX foci in irradiated cells. Both manual and automated quantification resulted in increasing focus numbers depending on dose. Comparison of automated reading with visual assessment for five manual observers resulted in a determination coefficient of R2 = 0.889. The inter-laboratory variability for five manual investigators of three laboratories was 38.4 %. Conclusion: The interpretation system AKLIDES® demonstrated a high correlation with visually observed results. High inter-laboratory variability found for manual investigations revealed the usefulness for a standardized technique for evaluation of γH2AX foci. © 2012 Informa UK, Ltd.


Grossmann K.,Lausitz University of Applied science | Grossmann K.,University of Leipzig | Roggenbuck D.,Medipan GmbH | Schroder C.,Lausitz University of Applied science | And 3 more authors.
Cytometry Part A | Year: 2011

Advances in immunofluorescence assay development paved the way for the simultaneous detection of several antibodies in one sample, for the serological diagnosis of systemic rheumatic diseases. Standardized automated screening of such antibodies can be achieved by HEp-2 cell-based indirect immunofluorescence (IIF) using a multicolor fluorescence imaging technical platform. To create a common platform for both screening and specific antibody assessment, multiplex measurement of antibodies using fluorescence-coded immobilized microbeads was employed on the same platform. The multicolor fluorescence detection system VideoScan (AKLIDES®) was used for the fluorescence analysis of a multiplex microbead-based immunoassay (MIA). First, immunoglobulin G (IgG) was covalently coupled to one microbead population in duplicate and in three independent experiments. The coupled IgG was detected by a Cy™5-conjugated secondary antibody. Thus, intra- and interassay coefficients of variation (CV) were obtained. Second, a multiplex determination of antinuclear autoantibodies (ANA) to Scl-70, Sm, dsDNA, SS-A (Ro60), CENP-B, and La/SS-B by solid-phase MIA was investigated, using 72 sera from patients with autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis (SS). The reproducibility study revealed intra-assay CVs ranging from 3.2% to 9.9%, and interassay CVs ranging from 9.6% to 14.7%. The detection of Scl-70-, Sm-, CENP-B-, and La/SS-B-ANA with MIA showed very good agreement with the ELISA results (kappa = 1.0). The resulting relative sensitivities and specificities for Scl-70-, Sm-, CENP-B-, dsDNA-, and La/SS-B-ANA were 100%, respectively, with the exception of dsDNA (specificity 97%). Multiplex detection by immobilized fluorescence-coded microbeads using multicolor fluorescence is a reliable method for the assessment of rheumatic-disease-specific antibodies. Multicolor fluorescence analyses with pattern detection algorithms provide a common platform technique for both the screening of ANA by cell-based IIF and specific antibody assessment by multiplex detection. © 2011 International Society for Advancement of Cytometry.


The invention relates to a method for automated determination of immunofluorescent foci by means of an immunofluorescence assay using synthetic calibration particles, in addition to a system and kit for carrying out the method. In a preferred embodiment the method is characterized in that the immunofluorescent foci are gamma H2Ax foci.


The invention relates to a method and device for disease diagnosis that simultaneously detects antibodies bound to cellular and/or tissue substrates and antibodies bound to synthetic substrates, such as microparticles or beads coated with specific antigens, thereby providing a one-step method for the simultaneous detection and characterization of disease-associated antibodies at both low (cellular and/or tissue) and high (antigen) specificity.

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