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.

Sciatic nerve lipoprotein lipase is reduced in streptozotocin-induced diabetes and corrected by insulin.

The metabolic abnormalities underlying the cause of diabetic neuropathy have been the subject of much debate. Lipoprotein lipase (LPL) is a 56-kDa enzyme produced by several tissues in the body and has recently been shown in vitro to be expressed in cultured Schwann cells, where it is important in phospholipid synthesis. This suggests a role for LPL in myelin biosynthesis in the peripheral nervous system. The aim of this study was to determine if acute streptozotocin (STZ)-induced diabetes reduces the expression and regulation of sciatic nerve LPL in vivo. Adult Sprague Dawley rats were rendered diabetic via an sc injection of STZ. A decrease in sciatic nerve LPL activity was observed in the STZ-treated rats after just 2 d of diabetes and remained significantly reduced for at least 35 d. The decrease in LPL activity coincided temporally with a drop in motor nerve conduction velocity. Treatment with insulin for 4 d showed a normalization of sciatic nerve LPL activity. These results show that STZ-induced diabetes causes a decrease in LPL activity in the sciatic nerve that, as in other tissues, is reversible with insulin treatment. These data may suggest a role for LPL in the pathophysiology of diabetic neuropathy.

A lipoprotein-containing particle is transferred from the serum across the mammary epithelium into the milk of lactating mice.

To investigate the role of low-density lipoprotein (LDL) in the delivery of cholesterol to the mammary gland during pregnancy and lactation, we examined the distribution of radioactivity from (125)I-tyramine cellobiose-LDL injected into the tail vein of female mice at various stages of the reproductive cycle. Changes in the proportion of isotope taken up by the mammary gland largely reflected the increased weight of the gland in pregnancy and lactation. In addition, during lactation, radioactivity was found in the milk and was associated with a protein of the molecular weight of apoB-100. Quantitatively similar results were obtained with mice homozygous for disruption of the LDL receptor gene (LDLR null). Analysis of endogenous lipoproteins showed that the milk lipoprotein particles were denser than the corresponding serum lipoproteins and largely depleted of triglyceride and cholesterol. Using fluorescence microscopy we visualize the sorting of apoB protein from the LDL lipid phase at the basal surface of the mammary epithelial cell of both wild-type and LDLR-null mice. Our findings provide evidence that the mammary epithelium of the lactating mouse is able to take up LDL from the plasma by a non-LDLR-mediated process. An apoB-containing particle from which the cholesterol has been removed is transferred into milk.

Increased intracellular triglyceride in C(2)C(12) muscle cells transfected with human lipoprotein lipase.

Much of the knowledge about the cell biology of lipoprotein lipase (LPL) in vitro has been gained from adipose tissue model systems. However, the importance of skeletal muscle lipoprotein lipase (SMLPL) to both lipoprotein and muscle metabolism remains unclear. Although the production of LPL in cultured myocytes has been documented, the amount of enzyme activity produced is small. To develop a more suitable tissue culture model for SMLPL, mouse C(2)C(12) myoblasts were stably transduced with a retroviral vector encoding the full-length human LPL (hLPL) cDNA. Control cells were transduced with a vector encoding beta-galactosidase. LPL expression was assayed as a function of cell growth by measuring LPL activity on days 3, 7, 9, 11, and 14 after subculture. The hLPL-transduced myoblasts increasingly overexpressed both heparin-releasable (HR) and intracellular (IN) LPL activity compared to nontransduced myoblasts (P < 0.001 at Day 11) and myoblasts transduced with the control vector (P < 0.001 at Day 11). This increase occurred while LPL mRNA levels remained stable between days 3 and 14. As expected, IN LPL activity was also increased in the transduced cells. High levels of LPL activity were also obtained after differentiating the C(2)C(12) cells into myotubes by serum deprivation. Additionally, throughout the time course, C(2)/LPL cells had greater amounts of intracellular triglyceride than both the C(2)C(12) and the C(2)/beta-GEO cells (P = 0.005 and P < 0.001, respectively) with the largest differences seen on day 14 of the time course (P = 0.001, C(2)/LPL vs C(2)C(12) (r) or C(2)/beta-GEO cells). Thus, C(2)C(12) myoblasts stably transduced with hLPL markedly overexpressed both HR and IN LPL activity compared to control cells which, in turn, was associated with increases in intracellular triglyceride content. Because LPL regulation in tissues is mostly posttranslational, this new in vitro model will permit the in-depth study of the posttranslational regulation of SMLPL and provide new insights into the fate of lipoprotein-derived fatty acids in muscle.

Lipoprotein lipase is expressed in cultured Schwann cells and functions in lipid synthesis and utilization.

We have previously demonstrated that lipoprotein lipase (LPL; triacylglycero-protein acylhydrolase, EC 3.1.1.34) is most likely expressed in the non-neuronal cells of the spinal cord, and glial cells may thus be the site of expression in the peripheral nervous system as well. We investigated the expression of LPL in cultured 1. 17 cells, an immortalized rat sciatic nerve Schwann cell line. The 1. 17 cells were shown to express LPL mRNA by reverse transcriptase-polymerase chain reaction analysis. The 1.17 Schwann cells demonstrated heparin-releasable lipolytic activity that was inhibited by the lipase inhibitor tetrahydrolipstatin in a dose-dependent manner. Preincubation of 1.17 cells with an anti-rat LPL antiserum reduced the heparin-releasable lipolytic activity to <10% of that measured in untreated cells. To investigate the role of LPL in Schwann cell lipid metabolism, 1.17 cells were incubated for up to 24 h with an emulsified [14C]triolein substrate and the incorporation of [14C]triolein radioactivity into various cellular lipids was examined in the presence of either anti-rat LPL antiserum or preimmune serum. Inhibiting LPL activity reduced the incorporation of 14C into cellular polar lipids, diacylglycerol, and cholesteryl esters by >80% at 2 and 6 h after addition of the radiolabeled substrate. At 24 h, radioactivity in diacylglycerol and cholesteryl esters was similar in cells treated with anti-LPL antiserum or preimmune serum, whereas 14C incorporation into polar lipids was still reduced by >60%. Separation of the polar lipids into individual lipid species revealed no specific changes in triolein-derived radioactivity incorporation across the phospholipid species examined. These results suggest that LPL-mediated hydrolysis of exogenous triacylglycerol is an important source of free fatty acids for the Schwann cell and thus may play a critical role in myelin biosynthesis in the peripheral nervous system.