Clinical and Analytical Impact of Moving from Jaffe to Enzymatic Serum Creatinine Methodology.

BACKGROUND: Identification and monitoring of chronic kidney disease (CKD) requires accurate quantification of serum creatinine. The poor specificity of Jaffe creatinine methods is well documented, and guidelines recommend enzymatic methodology. We describe our experience of moving from Jaffe to enzymatic creatinine methodology. We present comparison of >5000 paired Jaffe and enzymatic creatinine results, examine interferences, and attempt to assess clinical consequences of changing methodology. METHODS: Overall, 5303 serum samples received for routine creatinine measurement were analyzed using Jaffe and enzymatic methods with an Abbott Architect autoanalyzer. Associated results for glucose, total bilirubin, triglycerides, total protein, and hemolytic, icteric, and lipemic indexes were extracted from the laboratory database. CKD staging was estimated for each sample to assess potential clinical effects. RESULTS: The methods correlated well (r = 0.996) and showed good agreement (Passing-Bablok fit, y = 0.935x + 0.074). Paired analysis, however, showed significant differences (P < 0.001), and approximately 20% of results differed by more than ±10%. Multivariate analysis demonstrated independent associations between difference in creatinine results, glucose (P < 0.0001), and hemolytic index (P = 0.009). Glucose demonstrated positive interference in the Jaffe method, and hemolysis produced negative interference in the enzymatic method. Little or no association was observed with other analytes. CKD staging differed in 4% of samples. CONCLUSIONS: Differences between Jaffe and enzymatic serum creatinine results exceed the recommended 5% target for a significant proportion of samples, particularly at concentrations <1.13 mg/dL (100 µmol/L). Both glucose and hemolysis contribute to the variance in results. Although the clinical impact of these differences seems small, laboratories should continue moving toward enzymatic creatinine estimation to ensure the best estimate of renal function.

Clinical performance of the Roche cobas e411 automated assay system for thyrotropin-receptor antibodies for the diagnosis of Graves' disease.

BACKGROUND: The clinical performance of the Roche cobas e411 automated assay for the measurement of thyrotropin (TSH)-receptor antibodies (TRAbs) for the diagnosis of Graves' disease was evaluated in the setting of new referrals to a specialized thyroid clinic. METHODS: The final diagnosis of 102 new patients attending their first outpatient appointment at a thyroid clinic was correlated with the TRAbs result. In all cases, the diagnosis was made independently of the TRAbs result by the same consultant (ADT) based on clinical examination, thyroid function tests (TSH, free thyroxine, total triiodothyronine measured on Architect; Abbot Diagnostics), and a technetium-99m uptake and scan. TRAbs were measured using the cobas e411 (Roche Diagnostics). The clinical sensitivity and specificity of the assay were determined and compared with other published performance characteristics of the assay. RESULTS: Optimal sensitivity (95%) and specificity (98%) were obtained using a cut-off of 1.6 IU/L. The positive and negative predictive values at this cut-off were calculated as 98% and 94%, respectively. CONCLUSIONS: Using a cut-off of 1.6 IU/L, considered independently of thyroid function tests, the Roche cobas e411 automated immunoassay for TRAbs is a convenient, sensitive and specific tool for the differential diagnosis of hyperthyroidism.

Comparison of entropic contributions to binding in a "hydrophilic" versus "hydrophobic" ligand-protein interaction.

In the present study we characterize the thermodynamics of binding of histamine to recombinant histamine-binding protein (rRaHBP2), a member of the lipocalin family isolated from the brown-ear tick Rhipicephalus appendiculatus. The binding pocket of this protein contains a number of charged residues, consistent with histamine binding, and is thus a typical example of a "hydrophilic" binder. In contrast, a second member of the lipocalin family, the recombinant major urinary protein (rMUP), binds small hydrophobic ligands, with a similar overall entropy of binding in comparison with rRaHBP2. Having extensively studied ligand binding thermodynamics for rMUP previously, the data we obtained in the present study for HBP enables a comparison of the driving forces for binding between these classically distinct binding processes in terms of entropic contributions from ligand, protein, and solvent. In the case of rRaHBP2, we find favorable entropic contributions to binding from desolvation of the ligand; however, the overall entropy of binding is unfavorable due to a dominant unfavorable contribution arising from the loss of ligand degrees of freedom, together with the sequestration of solvent water molecules into the binding pocket in the complex. This contrasts with binding in rMUP where desolvation of the protein binding pocket makes a minor contribution to the overall entropy of binding given that the pocket is substantially desolvated prior to binding.

Residual ligand entropy in the binding of p-substituted benzenesulfonamide ligands to bovine carbonic anhydrase II.

In studies on the thermodynamics of ligand-protein interactions, it is often assumed that the configurational and conformational entropy of the ligand is zero in the bound state (i.e., the ligand is rigidly fixed in the binding pocket). However, there is little direct experimental evidence for this assumption, and in the case of binding of p-substituted benzenesulfonamide inhibitors to bovine carbonic anhydrase II (BCA II), the observed thermodynamic binding signature derived from isothermal titration calorimetry experiments leads indirectly to the conclusion that a considerable degree of residual entropy remains in the bound ligand. Specifically, the entropy of binding increases with glycine chain length n, and strong evidence exists that this thermodynamic signature is not driven by solvent reorganization. By use of heteronuclear (15)N NMR relaxation measurements in a series (n = 1-6) of (15)N-glycine-enriched ligands, we find that the observed thermodynamic binding signature cannot be explained by residual ligand dynamics in the bound state, but rather results from the indirect influence of ligand chain length on protein dynamics.

Large-scale modelling as a route to multiple surface comparisons of the CCP module family.

Numerous mammalian proteins are constructed from a limited repertoire of module-types. Proteins belonging to the regulators of complement activation family--crucial for ensuring a complement-mediated immune response is targeted against infectious agents--are composed solely of complement control protein (CCP) modules. In the current study, CCP module sequences were grouped to allow selection of the most appropriate experimentally determined structures to serve as templates in an automated large-scale structure modelling procedure. The resulting 135 individual CCP module models, valuable in their own right, are available at the online database http://www.bru.ed.ac.uk/~dinesh/ccp-db.html. Comparisons of surface properties within a particular family of modules should be more informative than sequence alignments alone. A comparison of surface electrostatic features was undertaken for the first 28 CCP modules of complement receptor type 1 (CR1). Assignments to clusters based on surface properties differ from assignments to clusters based on sequences. This observation might reflect adaptive evolution of surface-exposed residues involved in protein-protein interactions. This illustrative example of a multiple surface-comparison was indeed able to pinpoint functional sites in CR1.