[Improved patient safety through a clinical decision support system in laboratory medicine]. / Verbesserte Patientensicherheit durch "clinical decision support systems" in der Labormedizin.

BACKGROUND: Laboratory diagnostics are essential for diagnosis, initiation of therapy, and monitoring of patients. Laboratory results that are overlooked or incorrectly interpreted lead to adverse events and endanger patient safety. Clinical decision support systems (CDSSs) may facilitate appropriate interpretation of results and subsequent medical response. OBJECTIVES: The research project on digital laboratory medicine (AMPEL) aims at developing a CDSS based on laboratory diagnostics, which supports practitioners in ensuring the necessary medical consequences. MATERIALS AND METHODS: A literature review of CDSSs describes the current state of research. The research project AMPEL is presented with its objectives, challenges, and first results. Furthermore, the development of a framework and reporting system is illustrated through the clinical example of severe hypokalemia. RESULTS AND CONCLUSION: Through interdisciplinary development and constant optimization, a specific CDSS with high acceptance among clinicians was developed. Initial results in the case of severe hypokalemia show a positive effect on patient care. Thereby, more complex frameworks such as sepsis diagnostics or acute coronary syndrome are implemented. The limited availability of standardized and digital clinical data is challenging. In addition to the application of classic decision trees in CDSS, the use of machine learning offers a promising perspective for future developments.

Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein.

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.

Cloning and sequencing of cDNAs encoding the full-length mouse mannose 6-phosphate/insulin-like growth factor II receptor.

Overlapping clones encoding the complete coding sequence of the mouse mannose 6-phosphate/insulin-like growth factor II receptor have been isolated from a liver cDNA library and the nucleotide sequence has been determined. The open reading frame encodes a protein of 2482 amino acids that is 89 and 87% identical to the human and bovine receptors, respectively.

The effects of Mpl-ligand, interleukin-6 and interleukin-11 on megakaryocyte and platelet alpha-granule proteins.

Thrombopoietin (Mpl ligand), interleukin-6 (IL-6), and interleukin-11 (IL-11) differ in their effects on megakaryocyte maturation and development. In the present study, the impact of these thrombocytopoietic cytokines on biochemical and structural granule and membrane components was examined. Western blotting was performed on equivalent amounts of isolated megakaryocyte or platelet protein and the relative intensities of the enhanced chemiluminescent-visualized bands were quantitated by densitometry. Megakaryocyte growth and development factor (MGDF), a recombinant thrombopoietin-related molecule, significantly increased megakaryocyte fibronectin (72%), thrombospondin (55%), von Willebrand factor (28%) and p-selectin (CD62p) (37%) when compared to equivalent amounts of protein from saline-treated controls (p<0.01). Megakaryocyte fibrinogen was not increased. Ultrastructurally, there was a marked increase in ribosomes and rough endoplasmic reticulum even in mature-appearing megakaryocytes. Platelets from MGDF-treated mice showed small increases in fibronectin (8%), and CD62p (18%), but did not show increases in other measured alpha-granule proteins. Neither IL-6 nor IL-11 increased megakaryocyte or platelet alpha-granule proteins over levels observed in saline controls. IL-11 treated megakaryocytes, while also exhibiting an increase in ribosomes, were characterized by prominent cytoplasmic fragmentation. The study demonstrates the impact of Mpl ligand in increasing synthesized megakaryocyte alpha-granule proteins and of IL-11 in promoting megakaryocyte fragmentation.

Regulation of Corn Leaf Nitrate Reductase : II. Synthesis and Turnover of the Enzyme's Activity and Protein.

The appearance and disappearance of NADH:nitrate reductase (NR) in the leaves of corn (Zea mays L. W64A x W182E) were studied using activity assays, an enzyme-linked immunosorbent assay (ELISA) and western blotting. N-starved, etiolated corn plants were treated with nutrients containing either 35 millimolar NH(4)-nitrate or K-nitrate and immediately thereafter given light. The curve for enhancement of NR activity had three phases: 1 hour lag, 5 hour rapid increase, and steady state. The pattern for NR protein, as measured with the ELISA, also had three phases, but the increase was more rapid and the steady state was established earlier. To differentiate the effects of N nutrition from those of light, N-starved etiolated plants were given N nutrients 4 hours before light. During the dark pretreatment, NR activity and protein increased. When the light was turned on the NR activity and protein increased very rapidly without a lag. Western blots of polyacrylamide gels of native and denatured crude extracts showed that NADH:NR polypeptide was absent prior to treatment with N nutrients, but appeared after nitrate was given in dark or light. A low level of NR activity was found in N-starved, etiolated plants and it was shown by western blotting to be an NR form with a different electrophoretic mobility in nondenaturing gels. Since this minor NR form was not influenced by either nitrate or light, it was designated a constitutive NR. Dark decay of NR activity and protein was also studied. After the plants which had been in light with N nutrients for 24 hours were transferred to dark, the NR activity dropped by 30% within 1 hour, but the NR protein did not decrease. This inactivation of NR was further supported by returning the plants to the light after 1.5 hours of dark and finding the activity restored without change in NR protein. After the initial activity drop, a parallel decrease in NR activity and protein was observed, which was likely due to irreversible degradation by proteolysis.

Regulation of Corn Leaf Nitrate Reductase : I. Immunochemical Methods for Analysis of the Enzyme's Protein Component.

NADH:nitrate reductase was extracted from corn leaves (Zea mays L. W64A x W182E) and purified on blue Sepharose. After the nitrate reductase was further purified by polyacrylamide gel electrophoresis, it was used to immunize mice and a rabbit. Western blots of crude leaf extracts were used to demonstrate monospecificity of the mouse ascitic fluids and the rabbit antiserum. The electrophoretic properties of purified corn and squash NADH:nitrate reductases in both native and denatured states were shown to be similar using western blotting with mouse ascitic fluid. The corn leaf enzyme has a 115,000 polypeptide subunit like that of squash. Western blots could detect 3 to 10 nanograms of nitrate reductase protein. But the detection of proteolytic degradation products using western blotting was inconsistent and remains to be established. An enzyme-linked immunosorbent assay (ELISA) was developed for quantifying nitrate reductase protein in the crude extracts of corn leaves. Using a standard curve based on nitrate reductase activity, the ELISA for corn nitrate reductase could detect 0.5 to 10 nanograms of nitrate reductase protein and was adequately sensitive for quantitative analysis of nitrate reductase in crude extracts of leaves even when activity levels were very low. When the ELISA was used to compare the nitrate reductase protein content of corn roots and leaves, these tissues were estimated to contain 0.24 to 0.5 and 4 to 5 micrograms nitrate reductase protein/gram root and leaf, respectively.

A method to increase efficiency and minimize anomalous electrophoretic transfer in protein blotting.

We find that in protein blotting, the electrophoretic transfer efficiency of a given protein can be markedly affected by other components in the sample. Using a variety of electrophoretic elution protocols, we obtained very inefficient transfer of purified 125I-labeled 275-kDa cation-independent mannose 6-phosphate receptor from a sodium dodecyl sulfate-polyacrylamide gel to a membrane. Surprisingly, the transfer efficiency increased significantly if other proteins that migrated in the vicinity of the receptor were present. This may be a generally unappreciated but potentially widespread phenomenon as other but not all radiolabeled proteins examined behaved similarly. This carrier effect occurs under conditions typically used in protein blotting experiments and can lead to large errors in quantitative analysis. We developed two methods to minimize the carrier effect. First, addition of excess carrier protein to samples prior to electrophoresis saturated the carrier effect so that different samples had similar transfer efficiencies. Second, addition of either bovine serum albumin or sodium deoxycholate to the cathodic electro-elution buffer markedly increased transfer efficiency of radiolabeled proteins that exhibited poor transfer efficiency and minimized transfer differences between samples that contained or lacked carrier. These methods should be generally applicable to standard protein blotting and analysis protocols.

Soluble insulin-like growth factor II/mannose 6-phosphate receptor carries multiple high molecular weight forms of insulin-like growth factor II in fetal bovine serum.

We have characterized a soluble form of the insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) and bound ligands from bovine serum. Fetal serum contained 2-8 mg/liter sIGF-II/MPR. Affinity-purified receptor isolated by adsorption to phosphomannan-agarose and elution with mannose 6-phosphate contained nearly stoichiometric amounts of bound 7.5-kDa IGF-II. In addition, at least 12 distinct 12-20-kDa proteins immunologically related to IGF-II also copurified with receptor. Receptor was separated from its associated ligands by acidification and gel filtration chromatography. Sequence analysis revealed that the 12-20-kDa proteins have the same amino termini as mature 7.5-kDa IGF-II. Protease and glycosidase treatments revealed that the different high molecular weight IGF-II species contain an identical COOH-terminal extension that is differentially glycosylated with O-linked sugars. Radiolabeled tracer experiments demonstrated that the sIGF-II/MPR carries approximately 1/4 of the IGF-II in fetal bovine serum. These results support a significant role for sIGF-II/MPR in the transport of circulating IGF-II isoforms during development.

Mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor. A consensus casein kinase II site followed by 2 leucines near the carboxyl terminus is important for intracellular targeting of lysosomal enzymes.

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) mediates intracellular sorting of lysosomal enzymes, binding lysosomal enzymes in the Golgi and delivering them to a lysosomal compartment. The receptor also mediates endocytosis of extracellular ligands. We have devised a new method that rigorously measures function of the CI-MPR in intracellular sorting and used it to identify a previously uncharacterized signal near the COOH terminus of the receptor that is required for sorting. We stably transfect mutant receptors into CI-MPR-deficient mouse L cells, isolate homogeneous clonal cell lines that express a range of receptor levels for each mutant, and assay each cell line for levels of receptor expression and secretion of total phosphorylated lysosomal enzymes. Examination of the secretion phenotype of the cells as a function of receptor levels provides a sensitive indicator of the intrinsic sorting efficiency of each mutant receptor. We find that chimeric CI-MPRs that contain the bovine extracytoplasmic domain and the human or mouse transmembrane and cytoplasmic domains function identically to the bovine receptor, thus demonstrating that sorting signals are conserved. Analysis of a series of truncation and alanine scanning mutants reveals that a consensus casein kinase II site followed by 2 leucines near the COOH terminus that has the sequence (-10)DDSDEDLL(-3) is important for receptor function in sorting of lysosomal enzymes.

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.

Intercellular localization of nitrate reductase in roots.

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO(3) (-) for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO(3) (-) likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO(3) (-), the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO(3) (-) occurs during uptake and reduction of exogenous NO(3) (-). Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO(3) (-). It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO(3) (-), (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO(3) (-) is taken up by cells of the cortex and stele, and/or (c) NO(3) (-) is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO(3) (-) reduction.