[Current aspects of quality assurance]. / Aktuelle Aspekte zur Qualitätssicherung.

The regulations for quality management in the rheumatology laboratory are demanding and on a qualitatively high level. They are the result of continuously more detailed quality requirements and their implementation. Not only the process of correctly measuring analytes but also aspects of preanalytics, postanalytics and interpretation of the results as well as validation of new test methods are becoming increasingly more important for the diagnostic process.

Is it necessary to split nitrogen fertilization for winter wheat? On-farm research on Luvisols in South-West Germany.

Mineral nitrogen (N) fertilization in cereals is commonly split into three or four applications. In order to simplify N fertilization, a single N application either broadcast or placed on the soil surface was compared to conventionally split fertilization for winter wheat (Triticum aestivum L.). The 4-year experiment (2007-2010) was performed using a participatory approach on farmers' fields on deep loamy soils (Luvisols) in South-West Germany. Grain yield and crude protein contents differed only slightly or not at all between treatments including different N fertilizer types (calcium ammonium nitrate, urea ammonium nitrate solution, urea) and application techniques (broadcast, placed). Furthermore, no differences were found for the yield components ears/m2 and thousand grain weight. Inorganic N in the soil profile after harvest was generally below 40 kg N/ha and did not differ between treatments. In the area where N was placed, mineral N was depleted during the vegetation period. At the experimental sites a single N application in the period between tillering and stem elongation was sufficient to achieve high yield and quality of winter wheat without increased risk of nitrate leaching. This finding was independent of the method of application or the type of fertilizer.

Effects of different forms and origins of oilseeds on dynamics of ruminal biohydrogenation of long-chain fatty acids in vitro.

Dietary unsaturated fatty acids (FA) are intensively hydrogenated in the rumen, resulting in reduced amount of poly-unsaturated fatty acids (PUFA) and accumulation of several biohydrogenation (BH) products. In this study, BH of PUFA originating from different oilseeds (linseed, soya beans, sunflower seed and rapeseed) present in crushed oilseeds or their free oils were assessed in vitro. The assay substrates were incubated in buffered rumen fluid for 0, 6, 12 and 24 h. After incubation, the FA pattern of the incubated samples was analysed using gas chromatography. Biohydrogenation is defined as disappearance of double bonds (DB) calculated from the contents of unsaturated FA. After 24-h incubation, the DB contents of all oilseeds were reduced (p < 0.001) by 40-60%. The reduction was higher (p < 0.001) for the crushed form compared with the oil form. In addition, linseed and sunflower seed known as oilseeds with high contents of linolenic acid C18:3 c9,12,15 (LNA) and linoleic acid C18:2 c9,12 (LA), respectively, showed a higher (p < 0.001) accumulation of the BH intermediates conjugated linoleic acid (CLA, isomer C18:2 c9t11) and vaccenic acid (C18:1 t11) for the crushed form, when compared with the oil. These results suggest an inherent effect of the physical form of the assay oilseeds on in vitro BH. Changes in FA pattern during BH in vitro can be attributed to both source and physical form of the assay oilseeds. However, further investigations are warranted to ensure whether the observed in vitro effects on ruminal BH can be confirmed in vivo.

Micronized fibres affect in vitro fermentation under normal buffered and osmotic stress conditions using porcine inocula.

In this in vitro study, the modified Hohenheim gas test was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting a standard pig diet with varying levels of crude protein (CP; 20, 24 and 28% CP), and supplemented with one of three fibre sources manufactured by micronization treatment. These were wheat envelopes (MWE), pea fibre (MPF) and lupine fibre (MLF). For comparison, inulin was used. As intestinal bacteria have to cope with varying osmotic conditions in their ecosystem, fermentation was performed under normal buffered and osmotic stress conditions. After 24 h of fermentation, total gas production and ammonia production were measured. In addition, the effect of MWE and inulin on short-chain fatty acid (SCFA) production and numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp., Enterobacteriaceae, Enterococcus spp., Clostridium cluster XIVa and Clostridium cluster IV, were determined using quantitative real-time PCR. Under normal buffered conditions, supplementation of MWE resulted in increased (p < 0.05) SCFA, acetic, propionic and valerianic acid production at CP levels of 20 and 28%. There was an increase (p < 0.05) in ammonia production for the micronized supplements, and for MWE an increased (p < 0.05) branched-chain proportion was observed, possibly due to higher availability of protein for fermentation which was released during the micronization process. Osmotic stress conditions reduced (p < 0.05) total gas as well as total SCFA, acetic and propionic acid production for all treatments, while cell counts were increased (p < 0.05) for Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. Under normal buffered conditions in combination with 24 and 28% CP levels, lactobacilli were increased for MWE, compared to inulin (p < 0.05). In conclusion, micronized supplements such as MWE may beneficially modulate pigs' intestinal microbiota by increasing SCFA production in addition to a selective proliferation of lactobacilli.

Effects of long-term supplementation of chestnut and valonea extracts on methane release, digestibility and nitrogen excretion in sheep.

The long-term effects of adding chestnut (CHE; Castanea sativa) and valonea (VAL; Quercus valonea) tannin-rich extracts to sheep feed were investigated. In Experiment 1, sheep (65 kg BW) were fed 842 g/day of a ryegrass-based hay. The control-treated animals (CON) received 464 g/day of concentrate, and tannin-treated animals received the same amount of concentrate additionally containing 20 g of the respective tannin-rich extract. Hay and concentrates were offered together in one meal. After the onset of treatment, methane release was measured in respiration chambers for 23.5-h intervals (nine times) in a 190-days period. Faeces and urine were collected three times (including once before the onset of the tannin treatment) to assess digestibility and urinary excretion of purine derivatives. Based on the results obtained from Experiment 1, a second experiment (Experiment 2) was initiated, in which the daily tannin dosage was almost doubled (from 0.9 (Experiment 1) to 1.7 g/kg BW0.75). With the exception of the dosage and duration of the treatment (85 days), Experiment 2 followed the same design as Experiment 1, with the same measurements. In an attempt to compare in vitro and in vivo effects of tannin supplementation, the same substrates and tannin treatments were examined in the Hohenheim gas test. In vitro methane production was not significantly different between treatments. None of the tannin-rich extract doses induced a reduction in methane in the sheep experiments. On the 1st day of tannin feeding in both experiments, tannin inclusion tended to decrease methane release, but this trend disappeared by day 14 in both experiments. In balance period 3 of Experiment 1, lower dry matter and organic matter digestibility was noted for tannin treatments. The digestibility of CP, but not NDF or ADF, was reduced in both experiments. A significant shift in N excretion from urine to faeces was observed for both tannin-rich extracts in both experiments, particularly in Experiment 2. In balance period 2 of Experiment 2, an increased intake of metabolisable energy for VAL was observed. The urinary excretion of purine derivatives was not significantly different between treatments, indicating that microbial protein synthesis was equal for all treatments. Thus, we concluded that both tannin-rich extracts temporary affect processes in the rumen but did not alter methane release over a longer period.

[From heparin to apixaban: anticoagulants cut both ways?]. / Vom Heparin zum Apixaban: Was bringen die "neuen Antikoagulanzien"?

BACKGROUND: Regarding anticoagulant therapies there has been a remarkable shift in recent years. The objective of this brief overview is to provide relevant information and guidelines on the advantages and disadvantages of novel anticoagulants addressing specifically the surgical disciplines. Hitherto, conventional anticoagulant therapy in patients with a high thrombosis risk was largely limited to heparins and vitamin-K antagonists (VKA). Their modes of action, the difficulties in managing VKAs (e.g., bridging therapy) and the risk of HIT (heparin-induced thrombocytopenia) associated with heparins are briefly discussed. Novel anticoagulants supposedly eliminate these obstacles. Fondaparinux (Arixtra®) is a fully synthetic pentasaccharide which acts like a heparin but has an increased half life. Fondaparinux has a diminished risk of HIT. However, no specific antidote is currently available for Fondaparinux. The novel oral anticoagulants (NOAC) dabigatran etexilat (Pradaxa®), rivaroxaban (Xarelto®) and apixaban (Eliquis®), also known as "direct" anticoagulants, act independently from antithrombin by inhibiting thrombin, as in the case of dabigatran, or by inhibiting factor Xa, as in the case of rivaroxaban and apixaban. It is assumed that they are suitable for long-term use and do not require laboratory monitoring. Nevertheless, clinical experience is very limited and caution rather than quick conclusions is necessary. Two major drawbacks are on the one hand the risk of drug accumulation in kidney and/or liver disease and, on the other hand, the lack of specific antidotes. In addition, interactions with other medication may have unexpected effects on serum drug levels. Therefore, the analysis of drug levels in the plasma may become necessary in subgroups of patients. DISCUSSION AND CONCLUSION: Studies establishing clear recommendations for the desirable and measurable reference range are needed. Similarly, evidence-based recommendations regarding perioperative prevention of thrombosis are required ("bridging": yes or no?). Irrespective of these issues, the authors predict a further expansion of the use of NOACs.

Comparison of retention and prececal digestibility measurements in evaluating mineral phosphorus sources in broilers.

The objectives of this study were to compare measurements of retention and prececal (pc) digestibility in evaluating mineral phosphorus (P) sources in 3- and 5-wk-old broilers. A corn-soybean meal-based basal diet was used (0.35% P on DM basis). Anhydrous monosodium phosphate (MSP(a)) or anhydrous dibasic calcium phosphate (DCP(a)) was supplemented to increment the P concentration by 0.08%, 0.16%, and 0.24%. Titanium dioxide was used as the indigestible marker. Two retention trials with excreta collection from d 16 to 20 and d 30 to 34 were conducted (n = 8 birds per diet). Another 8 pens of 10 birds from the same hatch were allocated to each diet on d 11 or 25 each to measure pc digestibility in both age periods. After 10 d of feeding, these birds were euthanized and the content of a defined section of the terminal ileum was obtained. Percentage P retention and pc digestibility for MSP(a) and DCP(a) were calculated by linear regression analysis. In 3-wk-old broilers, P retention for MSP(a) was 70% and significantly higher (P < 0.001) than for DCP(a) (29%). Values determined for pc digestibility at the same age were very similar (67% for MSP(a) and 30% for DCP(a); P < 0.001). In 5-wk-old broilers, P retention was 63% (MSP(a)) and 29% (DCP(a); P < 0.001) and pc digestibility was 54% (MSP(a)) and 25% (DCP(a); P = 0.002). We concluded that both retention and pc digestibility can be used for evaluating mineral P sources in broilers based on a regression approach. In 3-wk-old broilers, results obtained with both approaches were the same. In 5-wk-old broilers, the ranking of the 2 P sources was also the same for both approaches. Values did not differ significantly between the 2 age periods, but further studies on the relevance of broilers' age in P evaluation are suggested.

Comparison and evaluation of bone measurements for the assessment of mineral phosphorus sources in broilers.

The main objective of this study was to compare different bone measurements in response to supplements of mineral P sources. Comparisons were also made with P retention and digestibility responses determined in a companion study and with blood inorganic phosphate (P(i)) responses. A corn-soybean meal-based basal diet was used (0.35% total P on DM basis). Anhydrous monosodium phosphate (MSP(a)) or anhydrous dibasic calcium phosphate (DCP(a)) was supplemented to increment the P concentration by 0.08%, 0.16%, or 0.24%. Each of the 7 diets was fed for 10 d starting 11 d (period 1) or 25 d posthatch (period 2). Bone ash and P were determined, and density criteria were measured using quantitative computed tomography. Responses were evaluated and compared based on linear regression analysis. In general, responses to MSP(a) had a greater slope than DCP(a) for all criteria studied. In period 1, differences between the slopes were significant (P < 0.05) for almost all bone criteria. In period 2, the slopes significantly differed for the amounts of ash and P of all bones studied, for tibia, tarsometatarus, and foot ash percentage, for total and cortical density of tibiae, but not for the other criteria. For the different bones, the ratio of slopes for MSP(a) and DCP(a) was very similar based on the amount of ash in both periods. Foot ash proved to be as sensitive as tibia ash for evaluation of mineral P sources in both periods. Ninety-four percent of the variance of the corticalis content based on quantitative computed tomography measurements could be explained by the amount of tibia ash in period 1. Blood serum P(i) and BW gain were not suitable for P evaluation. We concluded that the ranking of mineral P sources based on bone criteria differed from the ranking that was based on P retention or prececal digestibility. This underlines the need for developing a standard protocol of determination of available P in poultry.

Autoantibodies against inosine-5'-monophosphate dehydrogenase 2--characteristics and prevalence in patients with HCV-infection.

BACKGROUND: Indirect immunofluorescence (IIFT) on in house HEp-2 cell preparations revealed a novel antibody giving a granular cytoplasmic pattern not described before, which on two commercial cell preparations revealed a "rings and rods" pattern. This pattern was also observed in four HCV-RNA carriers and prompted the identification of the reactive antigen and the evaluation of the antibody prevalence in HCV-RNA carriers and control groups. METHODS: The antigen's molecular weight was determined by radioimmunoprecipitation of 35S-methionine labeled cell proteins. Expression library screening and sequencing was performed by standard techniques using an oligo(dT)-primed human HeLa cell cDNA expression library. Antibodies against the novel antigen Inositol-5'-monophosphatdehydrogenase 2 (IMPDH2) were analyzed by IIFT, western blot, line blot, and radioimmunoprecipitation assay (RIPA). IIFT was performed on commercial HEp-2 cells and cells cultivated in house for 24 - 60 hours, with or without the IMPDH2 inhibitors mycophenolic acid (MPA) or ribavirin, and subjected to various fixation conditions. Western and line blots were performed with IMPDH2 synthesized in E. coli, RIPA with 35S-methionine-IMPDH2 from in vitro transcription/translation products. Sera screened were positive for HCV-RNA (108), HBV-DNA (100), anti-mitochondrial (31), anti-actin (42), and anti-nuclear antibodies (51) and negative for HCV-RNA (100) and blood donors (100). RESULTS: IMPDH2 is capable of considerable intracellular rearrangements (upon action of inhibitors like MPA and ribavirin), which explains the contrasting immunofluorescence patterns in cells from different sources. By RIPA, proven to be the sole assay suitable for screening of anti-IMPDH2 in human sera, autoantibodies were found in 35.2% of HCV-RNA carriers and in low concentrations in 31% of anti-actin positive patients suspicious of autoimmune hepatitis. Antibodies reacted preferentially with conformational epitopes. Compared to the low concentration of anti-IMPDH2 found in other disease groups, high antibody concentrations were observed in HCV-RNA carriers. CONCLUSIONS: The common occurrence of anti-IMPDH2 in HCV-RNA carriers may be related to ribavirin therapy, causing intracellular aggregation of IMPDH2 thereby altering its immunogenicity. In this study the "rods and rings" immunofluorescence pattern observed could be ascribed to anti-IMPDH2. Anti-IMPDH2 may cause difficulties in interpretation of immunofluorescence patterns in routine autoantibody testing.

[Laboratory diagnosis of rheumatic diseases. Part 3: arthritides caused by infection]. / Labordiagnostik rheumatischer Erkrankungen: Teil 3: Infektbedingte Arthritiden.

This third part of this series of articles on laboratory diagnostics of rheumatic diseases considers the rheumatic diseases caused by infection by microorganisms, or reactive arthritides. The basis for laboratory diagnostics of infection-reactive arthritides is the investigation of anti-infection antibodies. In some situations, DNA amplification methods may be helpful. Bacterially infected joints should be immediately examined by arthrocentesis and microscopic examination and laboratory culture of the synovial fluid.

[Hereditary factor V deficiency and factor V inhibitor without bleeding. Rare causes of pathological screening tests of coagulation]. / Hereditärer Faktor-V-Mangel und Faktor-V-Inhibitor ohne Blutungsneigung. Seltene Ursachen pathologischer Globaltests.

Isolated reduction in factor V activity either occur in form of a hereditary deficiency of factor V as an acquired inhibitor against factor V. Diagnosis can not be made by bleeding alone because in both cases it can occur or not occur. Two patients were investigated showing pathological screening tests of coagulation without bleeding. A hereditary and an acquired deficiency of factor V were proved.

[Laboratory diagnostics of systemic autoimmune diseases. Part II: rheumatoid arthritis and vasculopathies]. / Labordiagnostik der systemischen Autoimmunerkrankungen. Teil II: Rheumatoide Arthritis und Vaskulopathien.

This is the second part in a series of articles on the laboratory diagnostics of rheumatic diseases. It addresses rheumatoid arthritis, systemic, anti-neutrophil cytoplasmatic antibody (ANCA) positive vasculitides and antiphospholipid-syndrome. The diagnostics of rheumatoid arthritis has been substantially improved by the recently introduced assay for antibodies against citrullinated peptides. In addition, a number of vasculitides can be differentiated by the presence of ANCA. Beta2-glycoprotein I antibodies and lupus anticoagulants are at present the most specific markers for antiphospholipid syndrome. Inflammatory activity can be monitored by determining the levels of acute phase proteins and erythrocyte sedimentation rate, but only in some situations by measuring immunoglobulins and interleukins.

[Laboratory diagnostics of systemic autoimmune diseases. Part 1. Collagenoses]. / Labordiagnostik der systemischen Autoimmunerkrankungen. Teil I: Kollagenosen.

This is the first part of a series of articles on the laboratory diagnostics of rheumatic diseases and will consider the systemic autoimmune diseases lupus erythematosus, Sjögren's syndrome, systemic sclerosis, dermato/polymyositis and mixed connective tissue disease (MCTD, SHARP syndrome). The basis for diagnostics is the presence of antinuclear antibodies (ANA). Initially, these antibodies are detected using a screening test. This must be followed by the identification of the patient's individual autoantibody specificities, which then yields important diagnostic clues. Disease activity may be monitored serologically by following the titers of selected autoantibodies and, in certain patients, by examining complement consumption.

Endothelial dysfunction in cold type complex regional pain syndrome.

The authors examined endothelial function in cold type chronic complex regional pain syndrome (CRPS) I using acetylcholine- and sodium nitroprusside-induced vasodilation combined with laser Doppler flowmetry in 14 patients and 10 controls. On the affected side, acetylcholine-induced vasodilation was significantly reduced in comparison to controls and the unaffected extremity. No significant differences were found after application of sodium nitroprusside. The results demonstrate impaired endothelial function in chronic CRPS I.

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.

Voltage and Ca(2+) dependence of pre-steady-state currents of the Na-Ca exchanger generated by Ca(2+) concentration jumps.

The Ca(2+) concentration and voltage dependence of the relaxation kinetics of the Na-Ca exchanger after a Ca(2+) concentration jump was measured in excised giant membrane patches from guinea pig heart. Ca(2+) concentration jumps on the cytoplasmic side were achieved by laser flash-induced photolysis of DM-nitrophen. In the Ca-Ca exchange mode a transient inward current is generated. The amplitude and the decay rate of the current saturate at concentrations >10 microM. The integrated current signal, i.e., the charge moved is fairly independent of the amount of Ca(2+) released. The amount of charge translocated increases at negative membrane potentials, whereas the decay rate constant shows no voltage dependence. It is suggested that Ca(2+) translocation occurs in at least four steps: intra- and extracellular Ca(2+) binding and two intramolecular transport steps. Saturation of the amplitude and of the relaxation of the current can be explained if the charge translocating reaction step is preceded by two nonelectrogenic steps: Ca(2+) binding and one conformational transition. Charge translocation in this mode is assigned to one additional conformational change which determines the equilibrium distribution of states. In the Na-Ca exchange mode, the stationary inward current depends on the cytoplasmic Ca(2+) concentration and voltage. The K(m) for Ca(2+) is 4 microM for guinea pig and 10 microM for rat myocytes. The amplitude of the pre-steady-state current and its relaxation saturate with increasing Ca(2+) concentrations. In this mode the relaxation is voltage dependent.

Peripheral auditory processing and investigations of the "precedence effect" which utilize successive transient stimuli.

This article addresses how a consideration of peripheral auditory processing can help to understand experiments concerning binaural precedence that employ successive binaural transients. It appears that much of the patterning of the behavioral data is amenable to an explanation based on peripheral interactions that result from auditory filtering and the functioning of auditory hair cells in combination with a binaural model based on cross correlation. A noteworthy aspect of this approach is that it does not include inhibitory mechanisms like those commonly invoked to explain binaural precedence.