Determination of lipase catalytic activity in two reference materials: BCR 693 and BCR 694 by titrimetry at constant pH.

Because routine assays for pancreatic lipase catalytic activity are not yet standardized, between-method comparability is very poor. This is mainly due to the lack of reference materials (RMs). The aim of this study was to assign values of catalytic concentration to two human pancreatic lipase RMs, one prepared from human pancreatic juice (BCR 693), the other obtained by recombinant technology (BCR 694). Lipase catalytic activity was assayed in five experienced laboratories, using aliquots from the same lot of triolein emulsion and a standardized titrimetric procedure, optimized with regard to substrate, cofactors and pH. The accepted sets of data (n = 4) gave a mean +/- the corresponding uncertainty expressed as the 0.95 confidence interval of 1732 +/- 72 U/l and 1043 +/- 60 U/l for BCR 693 and 694, respectively. Transferability of the whole operating procedure proved to be quite satisfactory. The authors conclude that both RMs can be used to verify the correct implementation of the standardized measurement procedure and to assign values to secondary lipase materials (commercial calibrators, control products) which, in turn, ensures traceability to the standardized procedure in this study, and contributes to the harmonization of laboratory results according to the Directive for in vitro Diagnostic Medical Devices.

Preparation and characterization of reference materials for human pancreatic lipase: BCR 693 (from human pancreatic juice) and BCR 694 (recombinant).

There is a lack of certified reference material (CRM) for lipase catalytic activity. Consequently between-method comparability is very poor. The aim of this study was to produce two lipase CRMs, one from human pancreatic juice (BCR 693), and another using recombinant technologies (BCR 694). Lipase was purified from pancreatic juice, using column chromatography and isoelectric focusing. Recombinant lipase was produced with a transfected cell line and purified with column chromatography. Adding buffered bovine serum albumin and subsequent freeze-drying were used to stabilize both materials. A standardized titrimetric method was employed to compare their catalytic properties to those of two plasma pools of patients suffering from acute pancreatitis. About 5 kU (titrimetry, 37 degrees C) of each material were obtained. They were lyophilized without apparent modifications of their catalytic properties, which stayed identical to those exhibited by the enzyme present in patient's pools. Stability of both materials was estimated at several years when stored in a dry form at -20 degrees C. Both materials appear to have similar catalytic properties and stability and were found commutable as regards a reference method and a routine measurement procedure. An international certification campaign will be carried out to assign values to BCR 693 and BCR 694.

On the mechanism of action of recombinant activated factor VII administered to patients with severe thrombocytopenia and life-threatening haemorrhage: focus on prothrombin activation.

We report the ex vivo effect of recombinant activated factor VII (rFVIIa) on prothrombin activation after whole blood clotting. Two patients with severe thrombocytopenia and life-threatening haemorrhage were successfully managed using a single dose of rFVIIa (90 microg/kg). Western blotting using antiprothrombin antibody showed that rFVIIa did not induce thrombin generation in citrated platelet-poor plasma. Patient sera showed significantly impaired prothrombin activation before and after rFVIIa administration. rFVIIa administration shortened the prothrombin time, activated partial thromboplastin time and Ivy bleeding time, and normalized the clot retraction. These data indicate that rFVIIa accelerated thrombin generation without significant increase of generated thrombin.