| 1. |
- Krisinger, Michael, et al.
(författare)
-
Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma.
- 2009
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 276, s. 6586-6602
-
Tidskriftsartikel (refereegranskat)abstract
- Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.
|
|
| 2. |
- Berntorp, Erik, et al.
(författare)
-
Standardization and clinical utility of thrombin-generation assays.
- 2008
-
Ingår i: Seminars in Thrombosis and Hemostasis. - : Georg Thieme Verlag KG. - 1098-9064 .- 0094-6176. ; 34:7, s. 670-682
-
Tidskriftsartikel (refereegranskat)abstract
- Thrombin generation is a key process that determines the extent of a hemostatic plug or a thrombotic process. The ensuing thrombin burst is crucial for the formation of a stable fibrin clot. During its active life, thrombin exerts a multitude of highly regulated actions on the blood and the vessel wall, including the clotting of fibrinogen. The inappropriate generation of thrombin may lead to pathologic processes, foremost of which are hemorrhagic or thrombotic diseases. The coagulation system is usually investigated by means of two in vitro classic clotting tests, the activated partial thromboplastin time (APTT) and prothrombin time (PT), which assess only time to initiation of clot formation and do not entirely reflect global hemostatic balance. The APTT and PT permit identification of connectivity between the component activities identified as required for plasma coagulation and define the concept of intrinsic and extrinsic coagulation pathways, which converge at the point of formation of the prothrombinase complex. However, the mechanisms established by in vitro tests are not always mirrored in the human pathologies associated with bleeding or thrombosis. The recent development of newer tests based on the continuous registration of thrombin generation under in vitro conditions that mimic more closely what occurs in vivo prompt a reinvestigation of the balance between procoagulants and anticoagulants in patients with various hemostatic disorders. Thrombin-generation assays not only provide an overall assessment of hemostasis but also target potential extrahemostatic effects of the generated thrombin, a potent agonist of a multitude of cellular activation pathways. Moreover, estimation of an individual's thrombin-generation potential may correlate more closely with a hypercoagulable or hypocoagulable phenotype when compared with traditional coagulation tests. In this review, we discuss to what extent thrombin generation can be expected to reflect the clotting function of blood, the development and use of different thrombin-generation assay systems suitable for detecting changes in the kinetics of thrombin generation, and the clinical utility of thrombin generation.
|
|
| 3. |
- Lippi, Giuseppe, et al.
(författare)
-
Preanalytical quality improvement : from dream to reality
- 2011
-
Ingår i: Clinical Chemistry and Laboratory Medicine. - : Walter de Gruyter. - 1434-6621 .- 1437-4331. ; 49:7, s. 1113-1126
-
Tidskriftsartikel (refereegranskat)abstract
- Abstract Laboratory diagnostics (i.e., the total testing process) develops conventionally through a virtual loop, originally referred to as "the brain to brain cycle" by George Lundberg. Throughout this complex cycle, there is an inherent possibility that a mistake might occur. According to reliable data, preanalytical errors still account for nearly 60%-70% of all problems occurring in laboratory diagnostics, most of them attributable to mishandling procedures during collection, handling, preparing or storing the specimens. Although most of these would be "intercepted" before inappropriate reactions are taken, in nearly one fifth of the cases they can produce inappropriate investigations and unjustifiable increase in costs, while generating inappropriate clinical decisions and causing some unfortunate circumstances. Several steps have already been undertaken to increase awareness and establish a governance of this frequently overlooked aspect of the total testing process. Standardization and monitoring preanalytical variables is of foremost importance and is associated with the most efficient and well-organized laboratories, resulting in reduced operational costs and increased revenues. As such, this article is aimed at providing readers with significant updates on the total quality management of the preanalytical phase to endeavour further improvement for patient safety throughout this phase of the total testing process.
|
|
| 4. |
- Salvagno, Gian Luca, et al.
(författare)
-
Thrombin generation assays (TGAs)
- 2017
-
Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1646, s. 515-522
-
Bokkapitel (refereegranskat)abstract
- In recent years, there has been a revival of interest in a time-honored tool of the clotting trade: the thrombin generation curve or thrombin generation (TG) assay (TGA) as we now call it. Thrombin generation is a key coagulation process that determines the extent of a hemostatic plug or a thrombotic process. The recent development of newer tests based on the continuous registration of TG under in vitro conditions that mimic more closely what occurs in vivo prompted us to reinvestigate the balance between procoagulants and anticoagulants in patients. Measurement of TG is accepted as a research tool, but the variety of sources and concentrations of reagents, as well as technical constraints, limit its potential for proper clinical use. The newer TGAs not only provide an overall assessment of hemostasis but also provide promising laboratory tools for investigating hemorrhagic coagulopathies and monitoring replacement therapy in several clinical conditions.
|
|
| 5. |
- Salvagno, Gian Luca, et al.
(författare)
-
Thrombin Generation Testing for Monitoring Hemophilia Treatment: A Clinical Perspective
- 2010
-
Ingår i: Seminars in Thrombosis and Hemostasis. - : Georg Thieme Verlag KG. - 1098-9064 .- 0094-6176. ; 36:7, s. 780-790
-
Tidskriftsartikel (refereegranskat)abstract
- Thrombin generation is a key process that determines the extent of a hemostatic plug or a thrombotic process. The ensuing thrombin burst is crucial for the formation of a stable fibrin clot. During its active life, thrombin exerts a multitude of highly regulated actions on the blood and the vessel wall, among which is the clotting of fibrinogen. The inappropriate generation of thrombin may lead to pathological processes, foremost of which are hemorrhagic or thrombotic diseases. The coagulation system is usually investigated by means of two in vitro classical clotting tests, the activated partial thromboplastin time and prothrombin time. These assays assess only the time taken to form a clot and do not entirely reflect global hemostatic balance. They permit identification of connectivity between the component activities identified as required for plasma coagulation and define the concept of intrinsic and extrinsic coagulation pathways, which converge at the step of formation of the prothrombinase complex. However, the mechanisms established by in vitro tests are not always mirrored in the human pathologies associated with bleeding or thrombosis. The recent development of newer tests based on the continuous registration of thrombin generation (TG) under in vitro conditions that mimic more closely what occurs in vivo prompted us to reinvestigate the balance between procoagulants and anticoagulants in patients. Thrombin generation assays (TGA) not only provide an overall assessment of hemostasis, but they also target potential extrahemostatic effects of the generated thrombin, a potent agonist of a multitude of cellular activation pathways. Moreover, estimation of an individual's thrombin generation potential may correlate more closely with a hyper- or hypocoagulablc phenotype, compared with traditional coagulation tests. In this review, we discuss to what extent TG can be expected to reflect the clotting function of blood, the development and use of different TGA systems suitable for detecting changes in the kinetics of thrombin generation, and the test's clinical utility for patients with hemophilia or von Willebrand disease.
|
|
| 6. |
- Topic, Elizabeta, et al.
(författare)
-
How to assess the quality of your analytical method?
- 2015
-
Ingår i: Clinical Chemistry and Laboratory Medicine. - : WALTER DE GRUYTER GMBH. - 1434-6621 .- 1437-4331. ; 53:11, s. 1707-1718
-
Tidskriftsartikel (refereegranskat)abstract
- Laboratory medicine is amongst the fastest growing fields in medicine, crucial in diagnosis, support of prevention and in the monitoring of disease for individual patients and for the evaluation of treatment for populations of patients. Therefore, high quality and safety in laboratory testing has a prominent role in high-quality healthcare. Applied knowledge and competencies of professionals in laboratory medicine increases the clinical value of laboratory results by decreasing laboratory errors, increasing appropriate utilization of tests, and increasing cost effectiveness. This collective paper provides insights into how to validate the laboratory assays and assess the quality of methods. It is a synopsis of the lectures at the 15th European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Continuing Postgraduate Course in Clinical Chemistry and Laboratory Medicine entitled "How to assess the quality of your method?" (Zagreb, Croatia, 24-25 October 2015). The leading topics to be discussed include who, what and when to do in validation/verification of methods, verification of imprecision and bias, verification of reference intervals, verification of qualitative test procedures, verification of blood collection systems, comparability of results among methods and analytical systems, limit of detection, limit of quantification and limit of decision, how to assess the measurement uncertainty, the optimal use of Internal Quality Control and External Quality Assessment data, Six Sigma metrics, performance specifications, as well as biological variation. This article, which continues the annual tradition of collective papers from the EFLM continuing postgraduate courses in clinical chemistry and laboratory medicine, aims to provide further contributions by discussing the quality of laboratory methods and measurements and, at the same time, to offer continuing professional development to the attendees.
|
|
