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A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.

ABSTRACT: Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm(3)) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm(3)) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.

SUBMITTER: Parfitt GJ

PROVIDER: S-EPMC3534019 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.

Parfitt Geraint J GJ Xie Yilu Y Reid Korey M KM Dervillez Xavier X Brown Donald J DJ Jester James V JV

PloS one 20121231 12

Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm(3)) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm(3)) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through ...[more]

PMID: 23300899

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Project description:ObjectivesPolycystic liver disease (PLD) is characterized by growth of hepatic cysts, causing hepatomegaly. Disease severity is determined using total liver volume (TLV), which can be measured from computed tomography (CT). The gold standard is manual segmentation which is time-consuming and requires expert knowledge of the anatomy. This study aims to validate the commercially available semi-automatic MMWP (Multimodality Workplace) Volume tool for CT scans of PLD patients.MethodsWe included adult patients with one (n = 60) or two (n = 46) abdominal CT scans. Semi-automatic contouring was compared with manual segmentation, using comparison of observed volumes (cross-sectional) and growth (longitudinal), correlation coefficients (CC), and Bland-Altman analyses with bias and precision, defined as the mean difference and SD from this difference. Inter- and intra-reader variability were assessed using coefficients of variation (CV) and we assessed the time to perform both procedures.ResultsMedian TLV was 5292.2 mL (IQR 3141.4-7862.2 mL) at baseline. Cross-sectional analysis showed high correlation and low bias and precision between both methods (CC 0.998, bias 1.62%, precision 2.75%). Absolute volumes were slightly higher for semi-automatic segmentation (manual 5292.2 (3141.4-7862.2) versus semi-automatic 5432.8 (3071.9-7960.2) mL, difference 2.7%, p < 0.001). Longitudinal analysis demonstrated that semi-automatic segmentation accurately measures liver growth (CC 0.908, bias 0.23%, precision 4.04%). Inter- and intra-reader variability were small (2.19% and 0.66%) and comparable to manual segmentation (1.21% and 0.63%) (p = 0.26 and p = 0.37). Semi-automatic segmentation was faster than manual tracing (19 min versus 50 min, p = 0.009).ConclusionsSemi-automatic liver segmentation is a fast and accurate method to determine TLV and liver growth in PLD patients.Key points• Semi-automatic liver segmentation using the commercially available MMWP volume tool accurately determines total liver volume as well as liver growth over time in polycystic liver disease patients. • This method is considerably faster than manual segmentation through the use of Hounsfield unit settings. • We used a real-life CT set for the validation and showed that the semi-automatic tool measures accurately regardless of contrast used for the CT scan or not, presence of polycystic kidneys, liver volume, and previous invasive treatment for polycystic liver disease.

Project description:IntroductionComputed tomography perfusion (CTP) imaging has become an important tool in evaluating acute recanalization treatment candidates. Large clinical trials have successfully used RAPID automated imaging analysis software for quantifying ischemic core and penumbra, yet other commercially available software vendors are also on the market. We evaluated the possible difference in ischemic core and perfusion lesion volumes and the agreement rate of target mismatch between OLEA, MIStar, and Syngo.Via versus RAPID software in acute recanalization treatment candidates.Patients and methodsAll consecutive stroke-code patients with baseline CTP RAPID imaging at Helsinki University Hospital during 8/2018-9/2021 were included. Ischemic core was defined as cerebral blood flow <30% than the contralateral hemisphere and within the area of delay time (DT) >3s with MIStar. Perfusion lesion volume was defined as DT > 3 s (MIStar) and Tmax > 6 s with all other software. A perfusion mismatch ratio of ⩾1.8, a perfusion lesion volume of ⩾15 mL, and ischemic core <70 mL was defined as target mismatch. The mean pairwise differences of the core and perfusion lesion volumes between software were calculated using the Bland-Altman method and the agreement of target mismatch between software using the Pearson correlation.ResultsA total of 1606 patients had RAPID perfusion maps, 1222 of which had MIStar, 596 patients had OLEA, and 349 patients had Syngo.Via perfusion maps available. Each software was compared with simultaneously analyzed RAPID software. MIStar showed the smallest core difference compared with RAPID (-2 mL, confidence interval (CI) from -26 to 22), followed by OLEA (2 mL, CI from -33 to 38). Perfusion lesion volume differed least with MIStar (4 mL, CI from -62 to 71) in comparison with RAPID, followed by Syngo.Via (6 mL, CI from -94 to 106). MIStar had the best agreement rate with target mismatch of RAPID followed by OLEA and Syngo.Via.Discussion and conclusionComparison of RAPID with three other automated imaging analysis software showed variance in ischemic core and perfusion lesion volumes and in target mismatch.

Dataset Information

A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.

Publications

A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.

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