Albe C. Swanepoel

Qualitative scanning electron microscopy analysis of fibrin networks and platelet abnormalities in diabetes.

Diabetes is a condition defined by hyperglycaemia and these patients have a high risk of thrombosis. Previous research showed that ultrastructural changes in clot formation occur in patients in whom there are changes in the coagulation system due to, for example, an inflammatory condition. In the current study, the ultrastructures of platelets and fibrin networks were investigated in 25 diabetic patients. Plasma smears, with and without the addition of thrombin, were prepared. Results indicated that the fibrin network in all diabetic patients had thickened masses of thin, minor fibres over the major fibres, a profile typical of an inflammatory condition. A changed platelet membrane ultrastructure could also be observed in the diabetic patients that revealed typical apoptotic morphology, in whom membrane blebbing could be seen. It can, therefore, be concluded that in diabetic patients, the ultrastructure of fibrin networks show a typical systemic inflammatory profile, although platelets seem to be apoptotic.

A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.

Differences in fibrin fiber diameters in healthy individuals and thromboembolic ischemic stroke patients

Cerebrovascular disease is one of the leading causes of death and the cause of long-term adult disability. An important characteristic of thromboembolic ischemic stroke is a prothrombotic or hypercoagulable state and altered fibrin clot structure, whereas a resistance to fibrinolysis is also present. An expansive fibrin network is created when adding thrombin, and in stroke, the network appears thickened, netted and matted, compared with that of healthy individuals. Although this is clearly visible in micrographs of patients, there is a need to quantify the changes. The current study, therefore, investigates fibrin fiber diameters in stroke patients and compares it to healthy individuals. The fiber diameters were measured in nanometres, with University of Texas Health Science Center at San Antonio (UTHSCSA) Image Tool. A total of 100 measurements were done for each of the 12 patients in the healthy control group, and the same number of measurements was done for 12 stroke patients. These measurements were statistically analysed with NCSS 2007, using a significance level of 0.05. Normality was assessed with the Shapiro–Wilk W test and the thickest and thinnest fiber of each individual in the two groups was quantified and differences between groups were assessed with the Students t-test. Results showed that there is a statistical difference in fibrin fiber thickness during thromboembolic ischemic stroke. We conclude that the changed coagulation and hemostasis, typically associated with stroke, causes a statistically relevant change in fibrin thickness, and that this netted and matted network is more resistant to lyses.

Scanning electron microscopy of fibrin networks in rheumatoid arthritis: a qualitative analysis

Rheumatoid arthritis is a chronic inflammatory condition that affects mainly synovial joints and has an impact on approximately 1% of the Western population. The coagulation process is altered in this condition, and this is frequently complicated by thrombocytosis. Changes in fibrin morphology have been linked with inflammation, and this, in turn, plays an important role in thrombosis. Changes in the fibrin fiber formation cause the alterations observed in thrombus morphology. In the current study, the ultrastructure of platelets and fibrin networks was investigated to determine whether any morphological changes are present in these structures in patients suffering from rheumatoid arthritis. Six patients diagnosed with rheumatoid arthritis took part in this study, and their clot morphology was compared to that of control subjects. Citrated blood with and without the addition of thrombin was used. Results indicated that the fibrin networks in the arthritis patients formed thick, matted layers. This matted appearance is due to a changed ultrastructure of the minor, thin fibers. Also, in these patients, spontaneous networks were created without the addition of thrombin, which indicates an abnormal hemostatic protein functioning, and the latter is expressed as visible changes in ultrastructure.

Flow cytometric analysis of platelets type 2 diabetes mellitus reveals ‘angry’ platelets

BackgroundThe function of platelets have extended way beyond the horizon of haemostasis and thrombosis, and are recognised as active participants in vascular inflammation, as well as in prothrombotic complications of cardiovascular diseases. We describe and compare platelet function in type II diabetes (with and without cardiovascular manifestation) and healthy individuals using scanning electron microscopy and flow cytometry.MethodsThirty subjects were recruited per group and informed consent was obtained from all participants. Diabetic patients were recruited from the diabetic clinic of the Steve Biko Academic Hospital (South Africa). Blood samples were drawn from all participants so that platelet specific antigens were analyzed in citrated whole blood. The platelet parameters used in the study were platelet identifiers (CD41 and CD42) and markers of platelet activation (CD62 and CD63).ResultsResults show that, compared to healthy individuals, both diabetic groups showed a significant difference in both platelet identifiers (CD41-PE, CD42b-PE) as well as markers indicating platelet activation (CD62P-PE and CD63-PE).InterpretationThe flow cytometric data shows that the platelet surface receptors and platelet activation are statistically elevated. This is suggestive of enhanced platelet activation and it appears as if platelets are displaying ‘angry’ behaviour. The lysosomal granules may play a significant role in diabetes with cardiovascular complications. These results were confirmed by ultrastructural analysis.

Estrogen causes ultrastructural changes of fibrin networks during the menstrual cycle: A qualitative investigation

Hormonal fluctuations may influence fibrin structure. During the menstrual cycle, plasma fibrinogen levels change, mainly due to the variations of estrogen. Throughout the menstrual cycle estrogen levels peak twice, first during the mid‐follicular phase and then a lower second peak during the luteal phase.

Scanning electron microscopy analysis of erythrocytes in thromboembolic ischemic stroke

Introduction:  Erythrocytes play an important role in hemostasis and disease conditions. During ischemic stroke, erythrocytes undergo oxidative and proteolytic changes resulting in a changed cellular rheology.

Novel diagnostic and monitoring tools in stroke : an individualized patient-centered precision medicine approach

Central to the pathogenesis of ischaemic stroke are the normally protective processes of platelet adhesion and activation. Experimental evidence has shown that the ligand-receptor interactions in ischaemic stroke represent a thrombo-inflammatory cascade, which presents research opportunities into new treatment. However, as anti-platelet drugs have the potential to cause severe side effects in ischaemic stroke patients (as well as other vascular disease patients), it is important to carefully monitor the risk of bleeding and risk of thrombus in patients receiving treatment. Because thrombo-embolic ischaemic stroke is a major health issue, we suggest that the answer to adequate treatment is based on an individualized patient-centered approach, inline with the latest NIH precision medicine approach. A combination of viscoelastic methodologies may be used in a personalized patient-centered regime, including thromboelastography (TEG®) and the lesser used scanning electron microscopy approach (SEM). Thromboelastography provides a dynamic measure of clot formation, strength, and lysis, whereas SEM is a visual structural tool to study patient fibrin structure in great detail. Therefore, we consider the evidence for TEG® and SEM as unique means to confirm stroke diagnosis, screen at-risk patients, and monitor treatment efficacy. Here we argue that the current approach to stroke treatment needs to be restructured and new innovative thought patterns need to be applied, as even approved therapies require close patient monitoring to determine efficacy, match treatment regimens to each patients individual needs, and assess the risk of dangerous adverse effects. TEG® and SEM have the potential to be a useful tool and could potentially alter the clinical approach to managing ischaemic stroke. As envisaged in the NIH precision medicine approach, this will involve a number of role players and innovative new research ideas, with benefits that will ultimately only be realized in a few years. Therefore, with this ultimate goal in mind, we suggest that an individualized patient-orientated approach is now available and therefore already within our ability to use.

Blood clot parameters: Thromboelastography and scanning electron microscopy in research and clinical practice

BACKGROUND Clotting parameters are informative of overall haematological healthiness of an individual. Particularly, clotting parameters can be used as a measure of the degree of pathology of the coagulation system. Thromboelastography (TEG) is a well-known technique that is an important point-of-care method, as well as research method. Scanning electron microscopy (SEM) is a novel research method, but with possible clinical application. However, there are no clear standardized guidelines for TEG and SEM result interpretation. MATERIALS AND METHODS We have an extensive database of results from TEG of hypercoagulable, hypocoagulable and healthy whole blood (WB) and platelet poor plasma (PPP). These results were generated using citrated PPP or WB, followed by the addition of CaCl2, to initiate clot formation. We also have an extensive and comprehensive database of thousands of clot micrographs, prepared for SEM. We reanalysed all our data to compile a user-friendly guideline for TEG and SEM. We also discuss the effects of different storage times on both WB and PPP. RESULTS We provide a quick and informative guide that discusses each TEG parameter, in both WB and PPP. Increases or decreases in the various parameters are indicative of either hyper- or hypocoagulability. We also show how hypo- and hypercoagulable clots look like, compared with healthy clots, using SEM analysis of clots created by adding thrombin to PPP. CONCLUSION For optimal and speedy interpretation of a patients coagulation status, it is essential for the clinician to make an informed and precise decision regarding clotting propensity. We believe this guideline will add to the standardization of TEG parameters, and ultimately contribute to the treatment of patients. These guidelines will also allow researchers to standardize their data interpretations and ultimately allow for the use of a global and inclusive database that might be included in precision medicine approaches.

Viscoelasticity and Ultrastructure in Coagulation and Inflammation: Two Diverse Techniques, One Conclusion.

The process of blood clotting has been studied for centuries. A synopsis of current knowledge pertaining to haemostasis and the blood components, including platelets and fibrin networks which are closely involved in coagulation, are discussed. Special emphasis is placed on tissue factor (TF), calcium and thrombin since these components have been implicated in both the coagulation process and inflammation. Analysis of platelets and fibrin morphology indicate that calcium, tissue factor and thrombin at concentrations used during viscoelastic analysis (with thromboelastography or TEG) bring about alterations in platelet and fibrin network ultrastructure, which is similar to that seen in inflammation. Scanning electron microscopy indicated that, when investigating platelet structure in disease, addition of TF, calcium or thrombin will mask disease-induced alterations associated with platelet activation. Therefore, washed platelets without any additives is preferred for morphological analysis. Furthermore, morphological and viscoelastic analysis confirmed that thrombin activation is the preferred method of fibrin activation when investigating fibrin network ultrastructure.