Verification and Validation of Digital Pathology (Whole Slide Imaging) for Primary Histopathological Diagnosis: All Wales Experience.

AIMS: The study is aimed to verify Aperio AT2 scanner for reporting on the digital pathology platform (DP) and to validate the cohort of pathologists in the interpretation of DP for routine diagnostic histopathological services in Wales, United Kingdom. MATERIALS METHODS AND RESULTS: This was a large multicenter study involving seven hospitals across Wales and unique with 22 (largest number) pathologists participating. 7491 slides from 3001 cases were scanned on Leica Aperio AT2 scanner and reported on digital workstations with Leica software of e-slide manager. A senior pathology fellow compared DP reports with authorized reports on glass slide (GS). A panel of expert pathologists reviewed the discrepant cases under multiheader microscope to establish ground truth. 2745 out of 3001 (91%) cases showed complete concordance between DP and GS reports. Two hundred and fifty-six cases showed discrepancies in diagnosis, of which 170 (5.6%) were deemed of no clinical significance by the review panel. There were 86 (2.9%) clinically significant discrepancies in the diagnosis between DP and GS. The concordance was raised to 97.1% after discounting clinically insignificant discrepancies. Ground truth lay with DP in 28 out of 86 clinically significant discrepancies and with GS in 58 cases. Sensitivity of DP was 98.07% (confidence interval [CI] 97.57-98.56%); for GS was 99.07% (CI 98.72-99.41%). CONCLUSIONS: We concluded that Leica Aperio AT2 scanner produces adequate quality of images for routine histopathologic diagnosis. Pathologists were able to diagnose in DP with good concordance as with GS. STRENGTHS AND LIMITATIONS OF THIS STUDY: Strengths of this study - This was a prospective blind study. Different pathologists reported digital and glass arms at different times giving an ambience of real-time reporting. There was standardized use of software and hardware across Wales. A strong managerial support from efficiency through the technology group was a key factor for the implementation of the study. LIMITATIONS: This study did not include Cytopathology and in situ hybridization slides. Difficulty in achieving surgical pathology practise standardization across the whole country contributed to intra-observer variations.

Ontogeny of endogenous secretion of immunoreactive-thyrotropin releasing hormone by the human placenta.

We studied endogenous production of immunologically active TRH (ir-TRH) by human placenta throughout gestation. Fragments (20 g) of placentae obtained between 7 and 41 weeks' gestation were incubated in TC-199 media with or without TRH degrading enzyme inhibitors (1 mM of dithiothreitol, 200 microm O-phenanthroline, 0.8 mM EDTA) at 37 C for 24 h. TRH was quantitated by RIA. Release of ir-TRH was also studied in an in vitro model of dually-perfused isolated lobule of human term placenta. Cellular localization of TRH was performed by staining early-, mid- and late-gestation placentae with anti-TRH rabbit polyclonal antibody, using the indirect avidin-biotin complex immunoperoxidase method. TRH was produced by placental fragments from 7 to 41 weeks' gestation. Placental TRH secretion was maximal between 7-12 weeks gestation both in presence (655 +/- 79 pg/10 mg protein) and absence (423 +/- 75 pg/10 mg protein) of enzyme inhibitors. Secretion of TRH declined with increasing gestation both with (y = 779 - 15x; r = 0.90; P < 0.001; n = 15) and without (y = 525 - 12x; r = 0.87; P < 0.001; n = 15) enzyme inhibitors. In the perfusion experiments, endogenous TRH was released predominantly into the fetal circulation, and its concentration was markedly higher in the presence of enzyme inhibitors (146 +/- 27 vs. 34 +/- 7 pg/mL; P < 0.001). Immunostaining of chorionic villi localized TRH to the cytoplasm of the syncytial layer, and the intensity declined with advancing gestational age. These data suggest that immunoactive TRH is produced by human placenta begin at 7 weeks gestation, and production declines with gestational age.

Molecular organization of tight and adherens junctions in the human placental vascular tree.

Tight and adherens junctions are major determinants of endothelial integrity. Molecules present therein have been implicated in vascular permeability, stability of junctions, angiogenesis and intracellular signalling. Using immunofluorescence and confocal scanning microscopy, the adherens junctions (AJs) in human placental vessels were found to contain the entire cadherin-catenin complex predicted from in vitro studies. Vascular endothelial cadherin (VE-cadherin) clusters were co-localized with beta-catenin, an important signal transduction ligand, and with alpha-catenin, which is thought to link the complex to the peri-junctional actin. Antibodies to plakoglobin, a molecule shown to be a component of stable adherens junctions, revealed immunoreactivity in clefts of stromal villous vessels, but weak or negative immunoreactivity in intermediate and terminal villi. Tight junctional molecules demonstrated a differential surface expression. Within the same villous tree, arteries, veins and arterioles contained occludin but the exchange vessels in terminal villi were immunonegative. ZO-1, however, was present throughout. Ultrastructurally, there were no differences in frequency, position or dimension of tight junctions in these vessels. They showed a consistent 4 nm separation between outer membrane leaflets regardless of their location in the vascular tree. Occludin is not necessary for formation of tight junctions in the placenta; it may have an accessory role providing stability or added adhesiveness to tight junctions of large vessels. Its absence in terminal villous microvessels, along with the weak plakoglobin immunoreactivity in AJs, suggest that the junctions here are less stable. This may allow the increased plasticity necessary in terminal villi for continual growth, proliferation and solute exchange.

Effects of gestational diabetes on junctional adhesion molecules in human term placental vasculature.

AIMS/HYPOTHESIS: The aim of this study was to investigate whether gestational diabetes mellitus, which occurs in the microvascular remodelling phase of placental development, causes alterations in surface expression of tight and adherens junctional molecules involved in endothelial barrier function and angiogenesis. METHODS: Term placenta, delivered by elective Caesarian section, from normal pregnancy (n = 5) and those complicated by gestational diabetes (n = 5) were perfusion-fixed and analysed by indirect immunofluorescence and confocal scanning microscopy. Using systematic random sampling, the surface expression of endothelial junctional proteins and the relative incidences of immunostained vessels were compared between the two study groups. Total vessel lengths were measured by stereological techniques. RESULTS: The adherens junctional molecules, vascular-endothelial cadherin and beta-catenin, and the tight junctional molecules, occludin and zonula occludens-1 were localised to paracellular clefts in both study groups. The diabetic placentae showed pronounced reductions in the intensity of immunofluorescence and in the number of immuno-positive vessels. A corresponding statistically significant increase (from 19% to 56%) in the percentage of vessels showing junctional anti-phosphotyrosine immunoreactivity was found. The differences observed represented real changes in the absolute lengths of immunostained regions along the vessels. The stereological measurements failed to detect any statistically significant change in the combined length of fetal vessels in gestational diabetic placenta. CONCLUSION/INTERPRETATION: Our results suggest that even short duration diabetic insult, alters the surface expression of placental junctional proteins. This alteration could be mediated by the tyrosine-phosphorylation pathway. The changes suggest impaired barrier function rather than accelerated vascular growth.

Ultrastructure of the early human feto-maternal interface co-cultured in vitro.

BACKGROUND: The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8-12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.

Morphological interactions of human first trimester placental villi co-cultured with decidual explants.

Abnormalities of pregnancy such as pre-eclampsia and intrauterine growth retardation are characterized by shallow trophoblastic invasion of the placental bed, the precise molecular pathophysiology of which remains to be fully elucidated. An in-vitro model involving a co-culture of first trimester placental villi and decidua parietalis explants (of 8-12 weeks gestation) was developed and used to characterize the migration and local invasion of trophoblast cells. Trophoblast proliferation (confirmed by Ki-67 immunostaining), differentiation and loose attachment of placental villi to the underlying decidual epithelium or stroma occurred within the first 24 h of co-culture. This was followed by erosion of the syncytial layer of the placental villi and commencement of a progressive cytotrophoblast invasion after 48 h of co-culture, which continued until 120 h, when the experiments were terminated. E-cadherin was expressed at the interfaces between trophoblast cells within the villi, but expression of this adhesion molecule seemed to be down-regulated in the invasive trophoblast cells. Our results suggest that the model could be useful in investigating the factors that control early human placentation and the feto-maternal interface.

Vascular endothelial cadherin and beta-catenin in human fetoplacental vessels of pregnancies complicated by Type 1 diabetes: associations with angiogenesis and perturbed barrier function.

AIMS/HYPOTHESIS: Increased angiogenesis of fetoplacental vessels is a feature of pregnancies complicated by Type 1 diabetes mellitus, but the underlying molecular mechanisms are unknown. This investigation tests whether the diabetic maternal environment alters the phenotypic expression of placental vascular endothelial cadherin and beta-catenin, which have been implicated as key molecules in barrier formation and angiogenesis in the endothelium. METHODS: Term placental microvessels from normal pregnancies (n=8) and from those complicated by Type 1 diabetes (n=8) were perfused with 76-Mr dextran tracers (1 mg/ml) and subjected to immunocytochemistry, immunoblotting and microscopy. Junctional integrity, localisation and phosphorylation were investigated along with total protein levels of vascular endothelial cadherin, beta-catenin and vascular endothelial growth factor. Stereological sampling and estimation tools were used to quantify aspects of angiogenesis and endothelial proliferation. RESULTS: In the Type 1 diabetic placentae, junctional localisations of vascular endothelial cadherin and beta-catenin altered significantly, with more than 50% of microvessels showing complete loss of immunoreactivity and with no overall loss of total protein. Tracer leakage was associated with these vessels. There was a two- to three-fold increase in vessels showing junctional phospho-tyrosine immunoreactivity and hyperphosphorylated beta-catenin. Vascular endothelial growth factor levels were higher in these placentae. A four-fold increase in endothelial proliferation was observed, along with an increase in total length of capillaries without any change in luminal diameter. CONCLUSIONS/INTERPRETATION: Molecular perturbations of vascular endothelial cadherin and beta-catenin occur in fetoplacental vessels of pregnancies complicated by Type 1 diabetes. Phosphorylation and loss of these molecules from the adherens junctional domains may be influenced in part by the elevated levels of vascular endothelial growth factor in the placenta. Perturbations of the junctional proteins may explain the observed breach in barrier integrity and may contribute to the mechanisms that drive proliferation and increases in capillary length.