Dataset Information

Quantitative histological image analyses of reticulin fibers in a myelofibrotic mouse.

ABSTRACT: Bone marrow (BM) reticulin fibrosis (RF), revealed by silver staining of tissue sections, is associated with myeloproliferative neoplasms, while tools for quantitative assessment of reticulin deposition throughout a femur BM are still in need. Here, we present such a method, allowing via analysis of hundreds of composite images to identify a patchy nature of RF throughout the BM during disease progression in a mouse model of myelofibrosis. To this end, initial conversion of silver stained BM color images into binary images identified two limitations: variable color, owing to polychromatic staining of reticulin fibers, and variable background in different sections of the same batch, limiting application of the color deconvolution method, and use of constant threshold, respectively. By blind coding image identities, to allow for threshold input (still within a narrow range), and using shape filtering to further eliminate background we were able to quantitate RF in myelofibrotic Gata-1low (experimental) and wild type (control) mice as a function of animal age. Color images spanning the whole femur BM were batch-analyzed using ImageJ software, aided by our two newly added macros. The results show heterogeneous RF density in different areas of the marrow of Gata-1low mice, with degrees of heterogeneity reduced upon aging. This method can be applied uniformly across laboratories in studies assessing RF remodeling induced by aging or other conditions in animal models.

SUBMITTER: Lucero HA

PROVIDER: S-EPMC5172452 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Quantitative histological image analyses of reticulin fibers in a myelofibrotic mouse.

Lucero Hector A HA Patterson Shenia S Matsuura Shinobu S Ravid Katya K

Journal of biological methods 20161122 4

Bone marrow (BM) reticulin fibrosis (RF), revealed by silver staining of tissue sections, is associated with myeloproliferative neoplasms, while tools for quantitative assessment of reticulin deposition throughout a femur BM are still in need. Here, we present such a method, allowing <i>via</i> analysis of hundreds of composite images to identify a patchy nature of RF throughout the BM during disease progression in a mouse model of myelofibrosis. To this end, initial conversion of silver stained ...[more]

PMID: 28008415

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Project description:The accuracy of quantitative stereological analysis tools such as the (physical) disector method substantially depends on the precise determination of the thickness of the analyzed histological sections. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. However, the orthogonal re-embedding (ORE) technique is quite work- and time-intensive and may produce inaccurate section thickness measurement values due to unintentional slightly oblique (non-orthogonal) positioning of the re-embedded sample-section. Here, an improved ORE method is presented, allowing for determination of the factual section plane angle of the re-embedded section, and correction of measured section thickness values for oblique (non-orthogonal) sectioning. For this, the analyzed section is mounted flat on a foil of known thickness (calibration foil) and both the section and the calibration foil are then vertically (re-)embedded. The section angle of the re-embedded section is then calculated from the deviation of the measured section thickness of the calibration foil and its factual thickness, using basic geometry. To find a practicable, fast, and accurate alternative to ORE, the suitability of spectral reflectance (SR) measurement for determination of plastic section thicknesses was evaluated. Using a commercially available optical reflectometer (F20, Filmetrics®, USA), the thicknesses of 0.5 ?m thick semi-thin Epon (glycid ether)-sections and of 1-3 ?m thick plastic sections (glycolmethacrylate/ methylmethacrylate, GMA/MMA), as regularly used in physical disector analyses, could precisely be measured within few seconds. Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Our results prove the applicability of SR to efficiently provide accurate section thickness measurements as a prerequisite for reliable estimates of dependent quantitative stereological parameters.

Dataset Information

Quantitative histological image analyses of reticulin fibers in a myelofibrotic mouse.

Publications

Quantitative histological image analyses of reticulin fibers in a myelofibrotic mouse.

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