Laboratory evaluation of the Coasys® Plus C coagulation analyzer.

INTRODUCTION: The Coasys® Plus C (Behnk Elektronik, distributed by Roche Diagnostics) is a coagulation analyzer for small to midsize clinical chemistry laboratories. We performed a laboratory evaluation. MATERIALS AND METHODS: After a familiarization period the dead volume, carry-over, capacity, within-assay reproducibility and imprecision were determined for the tests aPTT (STA APTT en STA Cephascreen), PT (STA Neoplastin Plus, STA Neoplastin R), INR (Hepato Quick), fibrinogen (STA Fibrinogen), antithrombin (Antithrombin III) and D-Dimer (Tina-quant D-Dimer Gen.2). A precision test and patient comparison with a STA-R Evolution® system were performed for aPTT (STA Cephascreen), PT (STA Neoplastin R), fibrinogen, antithrombin and D-Dimer. RESULTS: No carry-over was detected. The analyzer performed on average 66 measurements per hour. Within-assay reproducibility (% with normal/abnormal/extreme result): STA APTT 2.4/1.7/2.7; aPTT Cephascreen 1.4/3.1/1.8; PT Neoplastin Plus 1.2/2.1/1.7; PT Neoplastin R 2.3/-/3.1; INR Hepato Quick 0.6/0.9/1.9; fibrinogen 4.8/3.0/4.5; antithrombin 1.3/4.1/3.8; D-Dimer 19.9/4.3/4.3. Total imprecision (% with control 1/control 2/human pooled plasma): STA APTT 3.1/1.4/2.1; APTT Cephascreen 2.3/2.6/2.0; PT Neoplastin Plus 3.3/1.3/4.1; PT Neoplastin R 3.7/4.0/3.5; INR Hepato Quick 1.5/4.3/1.4; fibrinogen 3.4/5.8/8.1; antithrombin 3.0/6.4/2.4; D-Dimer 4.6/2.2/30.3. The correlation between the Coasys® Plus C and the STA-R Evolution® was good for aPTT, PT, antithrombin and fibrinogen. Some differences were observed for extreme deviant results for fibrinogen. For the analysis of D-Dimer, no sufficient correlation was found between the two analyzers. CONCLUSIONS: The Coasys® Plus C analyzer is fast, easy to handle and safe for the personnel. Its analytical performance makes it suitable for use in a clinical chemistry laboratory.

Evolutionary conservation of the lipopolysaccharide binding site of ß2-glycoprotein I.

ß2-Glycoprotein I (ß2GPI) is a highly abundant plasma protein and the major antigen for autoantibodies in the antiphospholipid syndrome. Recently, we have described a novel function of ß2GPI as scavenger of lipopolysaccharide (LPS). With this in mind we investigated the conservation of ß2GPI in vertebrates and set out to identify the binding site of LPS within ß2GPI. The genome sequences of 42 species were surveyed. Surface plasmon resonance (SPR) was performed with peptides to characterise the binding site of ß2GPI for LPS. ß2GPI could be identified in most tested vertebrates with a high overall amino acid homology of 80% or more in mammals. SPR revealed that a synthesised peptide (LAFWKTDA) from domain V of ß2GPI was able to compete for binding of ß2GPI to LPS. The AFWKTDA sequence was completely conserved in all mammals. The peptide containing the LPS binding site attenuated the inhibition by ß2GPI in a cellular model of LPS-induced tissue factor expression. Other important sites, such as the binding site for anionic phospholipids and the antiphospholipid antibody binding epitope, were also preserved. ß2GPI is highly conserved across the animal kingdom, which suggests that the function of ß2GPI may be more important than anticipated.

ß2-glycoprotein I: a novel component of innate immunity.

Sepsis is a systemic host response to invasive infection by bacteria. Despite treatment with antibiotics, current mortality rates are in the range of 20%-25%, which makes sepsis the most important cause of death in intensive care. Gram-negative bacteria are a prominent cause of sepsis. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, plays a major role in activating the host's immune response by binding to monocytes and other cells. Several proteins are involved in neutralization and clearance of LPS from the bloodstream. Here, we provide evidence that ß2-glycoprotein I (ß2GPI) is a scavenger of LPS. In vitro, ß2GPI inhibited LPS-induced expression of tissue factor and IL-6 from monocytes and endothelial cells. Binding of ß2GPI to LPS caused a conformational change in ß2GPI that led to binding of the ß2GPI-LPS complex to monocytes and ultimately clearance of this complex. Furthermore, plasma levels of ß2GPI were inversely correlated with temperature rise and the response of inflammatory markers after a bolus injection of LPS in healthy individuals. Together, these observations provide evidence that ß2GPI is involved in the neutralization and clearance of LPS and identify ß2GPI as a component of innate immunity.

Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies in blood of patients with thrombosis or fetal loss. There is ample evidence that beta(2)-glycoprotein I (beta(2)GPI) is the major antigen for antiphospholipid antibodies. The autoantibodies recognize beta(2)GPI when bound to anionic surfaces and not in solution. We showed that beta(2)GPI can exist in at least 2 different conformations: a circular plasma conformation and an "activated" open conformation. We also showed that the closed, circular conformation is maintained by interaction between the first and fifth domain of beta(2)GPI. By changing pH and salt concentration, we were able to convert the conformation of beta(2)GPI from the closed to the open conformation and back. In the activated open conformation, a cryptic epitope in the first domain becomes exposed that enables patient antibodies to bind and form an antibody-beta(2)GPI complex. We also demonstrate that the open conformation of beta(2)GPI prolonged the activated partial thromboplastin time when added to normal plasma, whereas the activated partial thromboplastin time is further prolonged by addition of anti-beta(2)GPI antibodies. The conformational change of beta(2)GPI, and the influence of the autoantibodies may have important consequences for our understanding of the antiphospholipid syndrome.