Evaluation of an automated chromogenic substrate assay for the rapid determination of hirudin in plasma.

A fully mechanized chromogenic substrate assay method for the rapid and specific determination of recombinant hirudin (r-hirudin) in citrated plasma on clinical chemistry analyzers (Hitachi 911 and Cobas Mira) is described. In a first step, 12 microliters sample volume is mixed with the chromogenic substrate. Due to the almost immediate action of hirudin the inhibitory reaction and the cleavage of the substrate is started simultaneously when bovine thrombin is added in excess. This excludes interferences by antithrombin III or heparin cofactor II. The change in absorbance/min is recorded at 405 nm. The measuring range is about 0.2-4.0 mg/l r-hirudin on both analyzers. Precision is characterized by intraassay coefficients of variation between 0.63% and 2.78% on the Hitachi 911 and 1.51% and 7.84% on the Cobas Mira, respectively and interassay coefficients of variation of 3.57% to 9.15% (Hitachi 911) and 3.72% to 12.99% (Cobas Mira) for the same r-hirudin plasma concentrations. The described determination of r-hirudin correlates well with an enzyme linked immunosorbent assay method for r-hirudin (Hitachi 911: r = 0.964, y = 0.978x + 0.038, n = 323; Cobas Mira: r = 0.964, y = 0.959x-0.003, n = 323).

Laboratory control of minimal heparinization during haemodialysis in patients with a risk of haemorrhage.

For patients undergoing dialysis with a high risk of haemorrhage there is no standardized procedure for anticoagulation during extracorporeal circulation. Minimal heparinization with a dose equivalent to half that used for chronic haemodialysis was employed in 49 patients (125 haemodialyses) performed after operative interventions (83.3%), after haemorrhagic events (5.2%) and after invasive investigations (11.5%). Using a biocompatible membrane and a low molecular weight heparin (bolus dose 500-1300 U; continuous infusion 100-400 U) it was possible to complete haemodialysis in 74 cases (Group 0) without clots appearing in the venous bubble trap of the tubing system. In 30 cases (Group 1) only small clots were detected at the end of haemodialysis, and in 13 cases (Group 2) larger clots (exceeding a diameter of 1 cm) were found. In eight cases (Group 3) partial or complete clot formation occurred in the tubing. No haemorrhagic complications were observed. Anti-Xa activity, thrombin-antithrombin III complex (TAT) and D-dimer were determined before haemodialysis, 2 h after the start of haemodialysis and on completion of the procedure. The anti-Xa activities ranged between < 0.2 and 0.56 U/ml. In contrast, at 2 h there were significant differences (P < 0.05) in the TAT concentrations between Group 0 and the other groups, as well as between Group 1 and Group 2 and 3. Significant differences (P < 0.05) in D-dimer levels occurred only at the end of haemodialysis. Minimal heparinization in haemodialysis is a practicable alternative in patients with a high risk of haemorrhage and extended coagulation monitoring is helpful in adjusting heparin dosage.

Influence of blood sampling from venipunctures and catheter systems on serial determinations of prothrombin activation fragment 1 + 2 and thrombin-antithrombin III complex.

To evaluate the influence of different blood sampling techniques on test results of thrombin-antithrombin III complex (TAT) and prothrombin fragment 1 + 2 (F1 + 2) serial determinations were performed. In six groups of nonrandomized patients (ten patients each) the concentrations of the coagulation markers of blood samples from central catheters (internal jugular, caval, Shaldon, pulmonary artery) and peripheral cannulas (17G and 18G) were compared with those of blood samples obtained simultaneously from direct venipunctures of the contralateral arm. Medians and 25th-75th percentiles of TAT and F1 + 2 concentrations of plasmas obtained from central catheters were not different from those taken from venipunctures. When delta mean values (catheter - venipuncture) were calculated negative results were obtained, indicating lower concentrations measured from blood sampled through central catheters with the exception of blood that taken from Shaldon catheters. Only for TAT concentrations significantly were lower values measured in blood samples taken from internal jugular catheters when compared with blood samples obtained from direct venipunctures. Significantly higher TAT concentrations were determined in blood samples obtained from Shaldon catheters. For both coagulation markers correlations were found between concentrations in blood samples from central catheters and venipunctures. In blood samples taken from peripheral venous cannulas only F1 + 2 concentrations correlated with the concentrations found in samples from direct venipuncture. In contrast to F1 + 2, TAT concentrations measured from blood samples via peripheral cannulas were determined significantly higher than those taken from direct venipunctures.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of component C of the methylreductase system in Methanococcus voltae and Methanobacterium thermoautotrophicum.

Antibodies were raised against homogeneous preparations of component C of the methylreductase system from Methanococcus voltae and Methanobacterium thermoautotrophicum. Cells of these organisms were fixed with paraformaldehyde and/or glutaraldehyde, sectioned, and labeled with antibodies and colloidal gold-labeled protein A. In M. voltae the gold particles were predominantly located in the vicinity of the cytoplasmic membrane. In rare cases a similar result was obtained also with M. thermoautotrophicum. However, in all but a few of the ultrathin sections of this bacterium, the label was randomly distributed in the cell interior. If one assumes a reliable fixation of all cell components, these results would suggest that the two distantly related methanogens studied have distinctive patterns for the localization of component C. The results with M. voltae are in agreement with recent findings that the methylreductase system is involved in the generation of a proton-motive force at the membrane.

Nickel requirement and factor F430 content of methanogenic bacteria.

Methanobacterium thermoautotrophicum has been reported to require nickel for growth and to contain high concentrations of a nickel tetrapyrrole designated factor F430. In this communication it is shown that all methanogenic bacteria investigated incorporated nickel during growth and also synthesized factor F430. This was also true for Methanobrevibacter smithii, which is dependent on acetate as a carbon source, and for Methanosarcina barkeri growing on acetate or methanol as energy sources. Other bacteria, including Acetobacterium woodii and Clostridium thermoaceticum, contained no factor F430. It is further shown that two yellow nickel-containing degradation products were formed from factor F430 when heated at pH 7. This finding explains why several forms of factor F430 were found in methanogenic bacteria when a heat step was employed in the purification procedure.