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Real-time analysis of blood coagulation and fibrinolysis: new rheological and optical sensing techniques for diagnosis of haemostatic disorders.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The haemostatic system has a dual paradoxical function in the body. It should arrest bleeding whenever needed, but also keep the blood flowing in the circulatory system without any obstructing blood clots. The system is complex with maoy intertwined processes that interact to produce a fine-tuned regulation of the performance. In case of malfunction in this regulation there may be an excessive coagulation ability, thrombophilia, or bleeding tendency, haemophilia. These are common disorders in the Western societies and may be lethal. The long-term airo of this work is therefore to improve the laboratory diagnosis of haemostatic disorders, for thrombophilia in particular. To achieve this goal a global approach has been chosen, meaning that the environment in which a blood sample is analysed should mimic the physiology of the haemostatic system to better elucidate the overall situation in a particular individual. A first attempt to assess the susceptibility for tissue plasminogen activator induced lysis and coagulum structure in plasma as markers for deep vein thrombosis showed promising results with 47% abnormals among the DVT patients included in the study. To improve this assay new sensing techniques were needed, since one of the most important conditions included in a global assay is analysis of whole blood, i.e. blood with all types of blood cells present. Whole blood is opaque and excludes the traditional optical methods that have been used for coagulation analysis. Several candidate techniques have been identified and surface plasmon resonance (SPR), quartz crystal microbalance-dissipation (QCM-D), and free oscillation rheometry (FOR) have been evaluated for haemostatic studies in this thesis. SPR is an optical surface sensitive technique that has showed promising results for measurements in blood plasma during coagulation and fibrinolysis and for whole blood coagulation. The SPR responses were sensitive to treatments with heparin and oral anticoagulants, which are substances used to treat thrombosis. QCM-D that is sensitive to mass deposition and viscoelastic changes in the sample at the quartz crystal surface has been tested in combination with SPR and provided new information about the viscoelastic properties of the coagulum, although with similar sensing depth as SPR. The idea of combined sensing techniques was reconsidered and resulted in a combination of SPR and FOR for siroultaneous real-time measurements in a blood sample. FOR is bulk sensitive and probes rheological changes in the sample. The combination was applied in studies of plasma and whole blood coagulation as well as plasma fibrinolysis. Coagulation studies including chemical surface modifications by using thiol-based self-assembled monolayers were also attempted. Finally, the FOR/SPR combination was found to be sensitive to inhibition of platelet aggregation and blood cell shape changes iroplying that studies on the cellular component of the blood is possible. In conclusion, the combination of FOR and SPR is a promising sensing system for an improved global assay for haemostatic disorders.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 72 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 663
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25656Local ID: 10032ISBN: 91-7219-764-1 (print)OAI: oai:DiVA.org:liu-25656DiVA: diva2:246204
Public defence
2001-03-16, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-05Bibliographically approved
List of papers
1. Abnormalities in coagulum lysis and structure are associated with deep venous thrombosis
Open this publication in new window or tab >>Abnormalities in coagulum lysis and structure are associated with deep venous thrombosis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The present study aimed to investigate the relationship between deep venous thrombosis (DVT) and fibrinolytic susceptibility of plasma coagulum, including the possible role of this property in laboratory diagnosis. From 276 patients consecutively admitted to hospital for suspected deep venous thrombosis (DVT), 75 patients and 47 controls were selected. With certainty, the patients and controls either had or did not have DVT. Fibrinolytic susceptibility was assayed by reacting plasma with thromboplastin and tissue plasminogen activator and recording a nephelometric signal. Coagulation time (CT), coagulum lysis time (CLT) and maximal increase in coagulum light scatter (CLS) were determined. Increase in D-dimer levels caused by coagulum lysis was also determined. This was viewed as a fibrinogen measure. CL T and CLS were interpreted as measures of fibrinolytic susceptibility and coagulum structure, respectively. CL T and CLS for patients and controls differed, p<0.025 and p<0.001, respectively. Compared to 5% for controls, 24% and 43% of the patients showed CL T and CLS outside the reference range. High fibrinogen levels could not explain the findings, since these were normal in most patients with abnormal CL T and CLS. Abnormal coagulum lysis and abnormal coagulum structure were thus found to be associated with DVT. Possible laboratory diagnostic role of CL T and CLS was investigated with bivariate reference ranges that excluded 5% and 0.3%. These ranges excluded significantly (p<0.0001) more patients, 47% and 27%, respectively. Tests for abnormal fibrinolytic susceptibility and coagulum structure may thus have a role in laboratory diagnosis of thrombotic disorders.

Keyword
Clot lysis, fibrin gel network, fibrinolysis, thrombosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81002 (URN)
Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2012-09-05Bibliographically approved
2. Blood plasma coagulation studied by surface plasmon resonance
Open this publication in new window or tab >>Blood plasma coagulation studied by surface plasmon resonance
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2000 (English)In: Journal of Biomedical Optics, ISSN 1083-3668, Vol. 5, no 1, 51-55 p.Article in journal (Refereed) Published
Abstract [en]

A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real time as a function of thromboplastin and heparin concentrations. The response curves were analyzed by curve fitting to a sigmoid curve equation, followed by extraction of the time constant. Clotting activation by thromboplastin resulted in increased time constant, as compared to spontaneously clotted plasma, in a dose dependent way. Addition of heparin to the thromboplastin-activated plasma counteracted this effect. Atomic force microscopy (AFM) pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and the fiber thickness increased with decreased thromboplastin concentration. The physical reason for the SPR signal observed is ambiguous and is therefore discussed. However, the results summarized in the plots and the fibrin network properties observed by AFM correlate well with present common methods used to analyze blood coagulation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25064 (URN)10.1117/1.429968 (DOI)9493 (Local ID)9493 (Archive number)9493 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-05Bibliographically approved
3. Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay
Open this publication in new window or tab >>Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay
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1999 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 14, no 8-9, 671-682 p.Article in journal (Refereed) Published
Abstract [en]

It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25062 (URN)10.1016/S0956-5663(99)00050-0 (DOI)9491 (Local ID)9491 (Archive number)9491 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-05Bibliographically approved
4. Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation
Open this publication in new window or tab >>Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation
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2000 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 15, no 11-12, 605-613 p.Article in journal (Refereed) Published
Abstract [en]

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25061 (URN)10.1016/S0956-5663(00)00125-1 (DOI)9490 (Local ID)9490 (Archive number)9490 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-05Bibliographically approved
5. Surface plasmon resonance and free oscillation rheometry in combination: A new approach forstudies on haemostasis and biomaterials
Open this publication in new window or tab >>Surface plasmon resonance and free oscillation rheometry in combination: A new approach forstudies on haemostasis and biomaterials
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In haemostasis and biomaterial research it is important to be able to study biological processes at surfaces and in the bulk. Surface plasmon resonance (SPR) is sensitive to changes at surface and free oscillation rheometry (FOR) probes the bulk. The present work demonstrates the usefulness of the combination of the techniques for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma, as well as coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with a different appearance than for plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This result indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyland hydroxyl-terminated self-assembled monolayers on gold and gold surfaces regenerated after coagulation by degradation of adsorbed proteins with trypsin and SOS were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation was found to be low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated difficulties for the coagulum to attach to the surface. The combination of SPR and FOR may be suited for studies of cell-surface interactions, and may find applications in studies of blood cell defects in patients and testing of medical substances.

Keyword
Surface plasmon resonance, free oscillation rheometry, whole blood coagulation, fibrinolysis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81014 (URN)
Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2012-09-05Bibliographically approved
6. Comparative studies with surface plasmon resonance and free oscillation rheometry on the inhibition of platelets with cytochalasin E and monoclonal antibodies towards GPIIb/IIIa
Open this publication in new window or tab >>Comparative studies with surface plasmon resonance and free oscillation rheometry on the inhibition of platelets with cytochalasin E and monoclonal antibodies towards GPIIb/IIIa
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2002 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 17, no 9, 761-771 p.Article in journal (Refereed) Published
Abstract [en]

In the haemostatic system a multitude of processes are intertwined in fine-tuned interactions that arrest bleeding, keep the circulatory system open, and the blood flowing. The occurrence of both surface and bulk interactions adds an additional dimension of complexity. These insights have led to the belief that global overall procedures can inform on the likely behaviour of the system in health and disease. Two sensing procedures: surface plasmon resonance (SPR), which senses surface interactions, and free oscillation rheometry (FOR), which senses interactions within the bulk, have been combined and evaluated. The contribution of blood cells, mainly platelets, to the SPR and FOR signals was explored by simultaneous SPR and FOR measurement during native whole blood coagulation, accelerated via the platelets through addition of SFLLRN peptide and inhibition of platelet aggregation with abciximab (ReoPro®) and of shape change with cytochalasin E. The SPR technique was found to be sensitive to inhibition of blood cell functions such as adhesion to and spreading on surfaces, as well as platelet aggregation. SPR seemed not to be directly sensitive to fibrin polymerisation in coagulating whole blood. The FOR technique detected the coagulation as a bulk phenomenon, i.e. the gelation of the blood due to fibrin formation was detected. The combination of SPR and FOR may therefore be suitable for studies on blood cell functions during coagulation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25066 (URN)10.1016/S0956-5663(02)00049-0 (DOI)9495 (Local ID)9495 (Archive number)9495 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-09-05Bibliographically approved

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