ABSTRACT
Stain colour estimation is a prominent factor of the analysis pipeline in most of histology image processing algorithms. Providing a reliable and efficient stain colour deconvolution approach is fundamental for robust algorithm. In this paper, we propose a novel method for stain colour deconvolution of histology images. This approach statistically analyses the multi-resolutional representation of the image to separate the independent observations out of the correlated ones. We then estimate the stain mixing matrix using filtered uncorrelated data. We conducted an extensive set of experiments to compare the proposed method to the recent state of the art methods and demonstrate the robustness of this approach using three different datasets of scanned slides, prepared in different labs using different scanners.
Subject(s)
Algorithms , Color , Coloring Agents/pharmacokinetics , Histological Techniques/methods , Image Processing, Computer-Assisted/methods , Models, Statistical , Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Colonic Neoplasms/diagnosis , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Eosine Yellowish-(YS)/pharmacokinetics , Female , Hematoxylin/pharmacokinetics , Histological Techniques/statistics & numerical data , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Staining and Labeling/methods , Staining and Labeling/statistics & numerical dataABSTRACT
BACKGROUND: Reflux following intravitreal injection is a common phenomenon, but it is unknown how much, if any, medication is lost as a result. Reflux is known to be a combination of vitreous and the injected agent, but the relative composition is unknown. This article describes a novel method for the measurement of the volume and composition of reflux and presents data from porcine eyes. METHODS: Twenty porcine eyes were injected with 0.05 ml of dye at intraocular pressures (IOPs) of 15, 20, 25 and 30 mmHg (five eyes per subgroup). Reflux was captured on filter paper and the area of saturation and color intensity of the dye were digitally analyzed. Total refluxed volume and proportion of dye versus vitreous fluid were calculated from linear regression lines created from known standards. RESULTS: Average (median) total volume of reflux from all eyes was 1.19 µl (0.93 µl), volume of injected dye refluxed was 0.47 µl (0.11 µl) and composition of reflux was 20.8% dye (15.5%). Less than 1% of the injected dye was lost to reflux. There were no differences between IOP groups in the total volume refluxed, the total amount of dye refluxed, the average composition of the reflux or the amount of injected dye refluxed (df = 3 for all comparisons; p = 0.58, p = 0.51, p = 0.55, p = 0.51, respectively). CONCLUSIONS: This novel method allows for measurement of quantity and composition of reflux following intravitreal injection in vitro. While reflux occurs frequently, it is predominantly composed of vitreous, not the injected agent. In fact, <1% of the original injection was lost to reflux.
Subject(s)
Aqueous Humor/metabolism , Hematoxylin/administration & dosage , Intraocular Pressure/physiology , Ocular Hypertension/metabolism , Vitreous Body/metabolism , Animals , Coloring Agents/administration & dosage , Coloring Agents/pharmacokinetics , Disease Models, Animal , Hematoxylin/pharmacokinetics , Intravitreal Injections , Ocular Hypertension/diagnosis , Ocular Hypertension/physiopathology , Prospective Studies , SwineABSTRACT
The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.
Subject(s)
Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Coloring Agents/pharmacokinetics , Hematoxylin/pharmacokinetics , Staining and Labeling/methods , Cell Nucleus/chemistry , Coloring Agents/chemistry , Hematoxylin/chemistryABSTRACT
Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.
