Methods
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R. Runge 1 , M. Wendisch 1 , G. Wunderlich 1 , D.Roggenbuck 2 , R. Hiemann 3 , U. Kasten-Pisula 4 , K. Storch 5 , J. Kotzerke 1 1 Klinik und Poliklinik für Nuklearmedizin, Universitätsklinikum C.G. Carus an der TU Dresden, 2 GA Generic Assays GmbH, Dahlewitz 3 Bio- Chemie- und Verfahrenstechnik, Fachhochschule Lausitz, 4 Klinik und Poliklinik für Strahlenbiologie und Radioonkologie, Universitätsklinikum-Hamburg-Eppendorf 5 OncoRay, Zentrum für Strahlenforschung in der Onkologie, Medizinische Fakultät, TU Dresden The measurement of DNA double strand breaks (DNA-DSB) with gH2AX-immunofluorescence microscopy (gH2AX-IFM) is an established method of detecting DNA damage, following cellular exposure to ionizing radiation. The visual counting of the foci is time consuming as well as objective and subject to artefacts. Therefore there is interest in an automated method, which also allows for standardization of the assessment. With intelligent computer based pattern recognition, inter- and intra-laboratory variance can be reduced. The slides were prepared for laboratory internal, external and automated analysis through irradiation of PC Cl3 thyroid cells with the beta-emitter 188 Re (ITG, Munich) in a dosage range of 0-5 Gy (dose-point-kernels). Immidiately after irradiation the cells were fixed, permeabilized and incubated with primary and secondary antibodies (anti-phospho-histone H2A.X, Alexa Fluor 488). The slildes were prepared for 5-fold determination, and distributed to three laboratories as blind samples for visual counting. Laboratory 1: University Hospital Dresden, Nuclear Medicine, 3 investigators; Laboratory 2: University Hospital Hamburg-Eppendorf, 1 investigator; Laboratory 3: TU Dresden, OncoRay, 1 investigator). The automated measurement was performed with the Aklides®- System (Medipan, Dahlewitz). Methods Results Both the visual interpretations of the investigators (laboratories 1-3) as well as the automatic evaluation show a dose-dependent, linear increase of the foci count/cells following irradiation with 188 Re. The Aklides ® algorithms were optimized to focus on counting and size definition (cell nuclei, foci) based on digitized images from the three investigators of laboratory 1. The results of the three investigators of laboratory 1 showed low within- laboratory variation and a good correlation with the results of the evaluation with Aklides ® . With one of the external investigators (laboratory 3), a significantly higher number of foci/cells were counted with 2 Gy und 5 Gy in comparison to laboratory 1. The results of the second external investigator (laboratory 2) showed at 0-2 Gy a significant inter-laboratory variation and at 5 Gy a high level of agreement with laboratory 1. Conclusions Figure 1: Comparison of visual counts by (A) three investigators from laboratory 1 and (B) investigators of laboratories 1-3 with automated evaluation. Representation of foci/cells following irradiation of PC Cl3 cells with 188 Re, means and standard deviations. Figure 2: Immunofluorescence microscopic detection of double strand breaks in PCCl3 cells following irradiation with 1Gy 188 Re (D- F) as well as in unirradiated control cells( A-C). A/D: Nuclear staining with DAPI B/E: Representation of gH2AX-Foci with AlexaFluor488 C/F: Overlaying of the fluorescence images G: Results of automated evaluation C The aim of this study is the adaptation of the pattern recognition algorithms of an automated system, for the reproducible analysis of gH2AX-Foci. In addition the visual interpretations of five investigators are compared to the automated analysis of Aklides®. Visual and automatic interpretation of gH2AX immunfluorescence microscopy images after irradiation with open radionuclides The adaptation of the automatic evaluation of AKLIDES® to the visual assessment makes the automated evaluation of gH2AX-Foci possible. When comparing the results of different laboratories problems arise, due to objective (differing microscopes, technique) and subjective causes (expectations of the evaluator). To achive a reduction in inter-laboratory variation, further standardization is necessary. A comparative analysis based on stored images is planned, to minimize objective and subjective error. The Aklides® algorithms were optimized to focus on counting and size definition (cell nuclei, foci) on the basis of digitalized images from three investigators from laboratory 1. Specimens were taken systematically with a 60x objective (fluorescencemicroscope IX81, Olympus) in three z- levels at 1µm intervals. The evaluation of the foci and local intensities took place in the z-levels with measurements from the groups of statistical, morphometric and form-describing characteristics. Background and Motivation A B C D E F A B C D E F G G A 0 5 10 15 20 25 30 35 0 0,5 0,75 1,0 2,0 5,0 Dose [Gy] Foci/ cells Automated Aklides system Visual evaluation by laboratory 1 B 0 5 10 15 20 25 30 35 40 0 0,5 0,75 1,0 2,0 5,0 Dose [Gy] Foci/cells Labor atory1 Laboratory 1 Laboratory 1 Aklides Laboratory 2 Laboratory 3
description
A. 35. Automated Aklides system. 30. Visual evaluation by laboratory 1. 25. 20. Foci/cells. 15. 10. 5. 0. 0. 0,5. 0,75. 1,0. 2,0. 5,0. Dose [Gy]. B. 40. Laboratory 1. Labor atory1. Laboratory 1. 35. Aklides. Laboratory 3. Laboratory 2. 30. 25. Foci / cells. 20. 15. - PowerPoint PPT Presentation
Transcript of Methods
R. Runge1, M. Wendisch1, G. Wunderlich1, D.Roggenbuck2, R. Hiemann3, U. Kasten-Pisula4, K. Storch5, J. Kotzerke1
1Klinik und Poliklinik für Nuklearmedizin, Universitätsklinikum C.G. Carus an der TU Dresden, 2 GA Generic Assays GmbH, Dahlewitz3 Bio- Chemie- und Verfahrenstechnik, Fachhochschule Lausitz, 4Klinik und Poliklinik für Strahlenbiologie und Radioonkologie, Universitätsklinikum-Hamburg-Eppendorf
5 OncoRay, Zentrum für Strahlenforschung in der Onkologie, Medizinische Fakultät, TU Dresden
The measurement of DNA double strand breaks (DNA-DSB) with gH2AX-immunofluorescence microscopy (gH2AX-IFM) is an established method of detecting DNA damage, following cellular exposure to ionizing radiation. The visual counting of the foci is time consuming as well as objective and subject to artefacts. Therefore there is interest in an automated method, which also allows for standardization of the assessment. With intelligent computer based pattern recognition, inter- and intra-laboratory variance can be reduced.
The slides were prepared for laboratory internal, external and automated analysis through irradiation of PC Cl3 thyroid cells with the beta-emitter 188Re (ITG, Munich) in a dosage range of 0-5 Gy (dose-point-kernels). Immidiately after irradiation the cells were fixed, permeabilized and incubated with primary and secondary antibodies (anti-phospho-histone H2A.X, Alexa Fluor 488). The slildes were prepared for 5-fold determination, and distributed to three laboratories as blind samples for visual counting. Laboratory 1: University Hospital Dresden, Nuclear Medicine, 3 investigators; Laboratory 2: University Hospital Hamburg-Eppendorf, 1 investigator; Laboratory 3: TU Dresden, OncoRay, 1 investigator). The automated measurement was performed with the Aklides®-System (Medipan, Dahlewitz).
Methods
Results
Both the visual interpretations of the investigators (laboratories 1-3) as well as the automatic evaluation show a dose-dependent, linear increase of the foci count/cells following irradiation with188Re. The Aklides® algorithms were optimized to focus on counting and size definition (cell nuclei, foci) based on digitized images from the three investigators of laboratory 1. The results of the three investigators of laboratory 1 showed low within-laboratory variation and a good correlation with the results of the evaluation with Aklides®. With one of the external investigators (laboratory 3), a significantly higher number of foci/cells were counted with 2 Gy und 5 Gy in comparison to laboratory 1. The results of the second external investigator (laboratory 2) showed at 0-2 Gy a significant inter-laboratory variation and at 5 Gy a high level of agreement with laboratory 1.
Conclusions
Figure 1: Comparison of visual counts by (A) three investigators from laboratory 1 and (B) investigators of laboratories 1-3 with automated evaluation. Representation of foci/cells following irradiation of PC Cl3 cells with188Re, means and standard deviations.
Figure 2: Immunofluorescence microscopic detection of double strand breaks in PCCl3 cells following irradiation with 1Gy 188Re (D-F) as well as in unirradiated control cells( A-C).A/D: Nuclear staining with DAPIB/E: Representation of gH2AX-Foci with AlexaFluor488C/F: Overlaying of the fluorescence imagesG: Results of automated evaluation
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The aim of this study is the adaptation of the pattern recognition algorithms of an automated system, for the reproducible analysis of gH2AX-Foci. In addition the visual interpretations of five investigators are compared to the automated analysis of Aklides®.
Visual and automatic interpretation of gH2AX immunfluorescence microscopy images after irradiation with open radionuclides
The adaptation of the automatic evaluation of AKLIDES® to the visual assessment makes the automated evaluation of gH2AX-Foci possible. When comparing the results of different laboratories problems arise, due to objective (differing microscopes, technique) and subjective causes (expectations of the evaluator). To achive a reduction in inter-laboratory variation, further standardization is necessary. A comparative analysis based on stored images is planned, to minimize objective and subjective error.
The Aklides® algorithms were optimized to focus on counting and size definition (cell nuclei, foci) on the basis of digitalized images from three investigators from laboratory 1. Specimens were taken systematically with a 60x objective (fluorescencemicroscope IX81, Olympus) in three z-levels at 1µm intervals. The evaluation of the foci and local intensities took place in the z-levels with measurements from the groups of statistical, morphometric and form-describing characteristics.
Background and Motivation
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