Clot Microstructure Research Articles

An Investigation Into the Effects of In Vitro Dilution With Different Colloid Resuscitation Fluids on Clot Microstructure Formation.

Balancing the beneficial effects of resuscitation fluids against their detrimental effect on hemostasis is an important clinical issue. We aim to compare the in vitro effects of 3 different colloid resuscitation fluids (4.5% albumin, hydroxyethyl starch [Voluven 6%], and gelatin [Geloplasma]) on clot microstructure formation using a novel viscoelastic technique, the gel point. This novel hemorheologic technique measures the biophysical properties of the clot and provides an assessment of clot microstructure from its viscoelastic properties. Importantly, in contrast to many assays in routine clinical use, the measurement is performed using unadulterated whole blood in a near-patient setting and provides rapid assessment of coagulation. We hypothesized that different colloids will have a lesser or greater detrimental effect on clot microstructure formation when compared against each other. Healthy volunteers were recruited into the study (n = 104), and a 20-mL sample of whole blood was obtained. Each volunteer was assigned to 1 of the 3 fluids, and the sample was diluted to 1 of 5 different dilutions (baseline, 10%, 20%, 40%, and 60%). The blood was tested using the gel point technique, which measures clot mechanical strength and quantifies clot microstructure (df) at the incipient stages of fibrin formation. df and clot mechanical strength decrease with progressive dilution for all 3 fluids. A significant reduction in df from baseline was recorded at dilutions of 20% for albumin (P < .0001), 40% for starch (P < .0001), and 60% for gelatin (P < .0001). We also observed significant differences, in terms of df, when comparing the different types of colloid (P < .0001). We found that albumin dilution produced the largest changes in clot microstructure, providing the lowest values of df (= 1.41 ± 0.061 at 60% dilution) compared with starch (1.52 ± 0.081) and gelatin (1.58 ± 0.063). We show that dilution with all 3 fluids has a significant effect on coagulation at even relatively low dilution volumes (20% and 40%). Furthermore, we quantify, using a novel viscoelastic technique, how the physiochemical properties of the 3 colloids exert individual changes on clot microstructure.

The effect of sepsis and its inflammatory response on mechanical clot characteristics: a prospective observational study.

Sepsis and its progression are known to have a major influence on the coagulation system. Current coagulation tests are of limited use when assessing coagulation in sepsis patients. This study aims to assess the potential for a new functional biomarker of clot microstructure, fractal dimension, to identify changes in the mechanical properties of clot microstructure across the sepsis spectrum (sepsis, severe sepsis and septic shock). A total of 100 patients that presented acutely to a large teaching hospital were included in this prospective observational study (50 sepsis, 20 severe sepsis and 30 septic shock) against a matched control of 44 healthy volunteers. Fractal analysis was performed, as well as standard markers of coagulation, and six plasma markers of inflammation. Fractal dimension was significantly higher in the sepsis and severe sepsis groups than the healthy control (1.78±0.07 and 1.80±0.05, respectively vs 1.74±0.03) (p<0.001), indicating a significant increase in mechanical clot strength and elasticity consistent with a hypercoagulable state. Conversely, fractal dimension was significantly lower in septic shock (1.66±0.10, p<0.001), indicating a significant reduction in mechanical clot strength and functionality consistent with a hypocoagulable state. This corresponded with a significant increase in the inflammatory response. This study confirms that clot microstructure is significantly altered through the various stages of sepsis. Of particular importance was the marked change in clot development between severe sepsis and septic shock, which has not been previously reported.

Effects of exercise intensity on clot microstructure and mechanical properties in healthy individuals

BackgroundExercise is well established to lead to exercise-induced hypercoagulability, as demonstrated by kinetic coagulation markers. It remains unclear as to whether exercise-induces changes lead in clot development and increased polymerisation. Fractal dimension (df) has been shown to act as a marker of clot microstructure and mechanical properties, and may provide a more meaningful method of determining the relationship between exercise-induced hypercoagulability and potential clot development. Methodsdf was measured in 24 healthy individuals prior to, after 5min of submaximal exercise, following maximal exercise, 45min of passive recovery and following 60min of recovery. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. ResultsSignificantly increased df was observed following exercise, returning to resting values following 60min of recovery. The relationship between df and mature clot microstructure was confirmed by SEM: higher df was associated with dense clots formed of smaller fibrin fibres immediately following exercise compared to at rest. Conventional markers of coagulation confirmed findings of previous studies. ConclusionThis study demonstrates that df is a sensitive technique which quantifies the structure and properties of blood clots following exercise. In healthy individuals, the haemostatic balance between coagulation and fibrinolysis is maintained in equilibrium following exercise. In individuals with underlying vascular damage who participate in exercise, this equilibrium may be displaced and lead to enhanced clot formation and a prothrombotic state. df may therefore have the potential to not only quantify hypercoagulability, but may also be useful in screening these individuals.

PO-25 - FATCAT: an observational cohort study investigating fractal dimension (df) as a biomarker of thrombogenicity in cancer associated thrombosis during chemotherapy for lung cancer

Venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) and pulmonary embolism (PE) are common complications in patients with cancer, affecting up to 18% of patients. VTE risk is increased by surgery and disease progression, whilst chemotherapy further increases risk up to 7-fold compared to patients without cancer. VTE contributes significantly to morbidity and mortality in patients with cancer, and is the second most common cause of death. Lung cancer is well established to be high risk for VTE, with up to a 22-fold increase in VTE risk associated with this malignancy, and 12% incidence in a recent study of patients with lung cancer undergoing chemotherapy. Furthermore, platinum based chemotherapy agents used in treatment of lung cancer are further associated with increased VTE risk. Current risk assessment tools have little value in predicting VTE risk, but prophylactic anticoagulation of patients with cancer increases bleeding incidence and no overall survival benefit. There is therefore a need for a pragmatic test with which assesses coagulation in patients with cancer, and potentially predict VTE risk, leading to personalised management and targeted treatment. We have previously demonstrated that fractal dimension (df) is sensitive to changes in clot microstructure in patients with lung cancer, assessing global coagulation in these patients. Furthermore, df is significantly different in patients with extensive disease (stages 3&4), which conventional laboratory markers failed to identify. Given the increased risk of VTE associated with chemotherapy, FATCAT will aim to assess changes in df in a larger cohort of patients with lung cancer undergoing chemotherapy, quantifying changes in df and relating these to clinical outcome. This is a prospective observational cohort study investigating changes in df in patients with lung cancer undergoing chemotherapy. Patients will have a new diagnosis of cytologically or histologically confirmed lung cancer planned for chemotherapy and no history of previous cancer treatment, any thromboembolic / haemostatic disorders or be on anticoagulation. Following a power calculation, 300 patients will be recruited and followed up for 1 year. df, Doppler ultrasonography and standard coagulation markers will be performed on recruitment, at the mid point, and on completion of chemotherapy in line with standard diagnostic procedures i.e. CT scanning. The primary endpoint of the study will be VTE diagnosis, whilst secondary outcomes will determine the change in df during and after treatment with chemotherapy.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

The Effects of Temperature on Clot Microstructure and Strength in Healthy Volunteers.

Anesthesia, critical illness, and trauma are known to alter thermoregulation, which can potentially affect coagulation and clinical outcome. This in vitro preclinical study explores the relationship between temperature change and hemostasis using a recently validated viscoelastic technique. We hypothesize that temperature change will cause significant alterations in the microstructural properties of clot. We used a novel viscoelastic technique to identify the gel point of the blood. The gel point identifies the transition of the blood from a viscoelastic liquid to a viscoelastic solid state. Furthermore, identification of the gel point provides 3 related biomarkers: the elastic modulus at the gel point, which is a measure of clot elasticity; the time to the gel point (TGP), which is a measure of the time required to form the clot; and the fractal dimension of the clot at the gel point, df, which quantifies the microstructure of the clot. The gel point measurements were performed in vitro on whole blood samples from 136 healthy volunteers over a temperature range of 27°C to 43°C. There was a significant negative correlation between increases in temperature, from 27°C to 43°C, and TGP (r = -0.641, P < 0.0005). Conversely, significant positive correlations were observed for both the elastic modulus at the gel point (r = 0.513, P = 0.0008) and df (r = 0.777, P < 0.0005) across the range of 27°C to 43°C. When temperature was reduced below 37°C, significant reductions in df and TGP occurred at ≤32°C (Bonferroni-corrected P = 0.0093) and ≤29°C (Bonferroni-corrected P = 0.0317), respectively. No significant changes were observed when temperature was increased to >37°C. This study demonstrates that the gel point technique can identify alterations in clot microstructure because of changes in temperature. This was demonstrated in slower-forming clots with less structural complexity as temperature is decreased. We also found that significant changes in clot microstructure occurred when the temperature was ≤32°C.

Altered clot microstructure detected in obstructive sleep apnoea hypopnoea syndrome

Abnormal clot microstructure plays a pivotal role in the pathophysiology of thromboembolic diseases. Assessing the viscoelastic properties of clot microstructure using novel parameters, Time to Gel Point (TGP), Fractal Dimension (df) and clot elasticity (G׳GP) could explain the increased cardiovascular and thromboembolic events in patients with Obstructive Sleep Apnoea Hypopnea Syndrome (OSAHS). We wanted to compare TGP, df, and G׳GP and their diurnal variation in OSAHS and symptomatic comparators. thirty six patients attending a sleep disturbed breathing clinic with symptoms of OSAHS were recruited. TGP, df and G׳GP were measured alongside standard coagulation screening, thrombin generation assays, and platelet aggregometry at 16:00h and immediately after an in-patient sleep study at 07:30h. OSAHS group had significantly lower afternoon df than comparators (1.705±0.033 vs. 1.731±0.031, p<0.05). df showed diurnal variation and only in the OSAHS group, being significantly lower in the afternoon than morning (p<0.05). Diurnal changes in df correlated with 4% DR, even after controlling for BMI (r=0.37, p=0.02). The lower df in the afternoon in OSAHS suggests a partial compensatory change that may make up for other pro-clotting abnormalities/hypertension during the night. The change to the thrombotic tendency in the afternoon is biggest in severe OSAHS. df Shows promise as a new microstructural indicator for abnormal haemostasis in OSAHS.

The antihemostatic profile of vitamin C: Mechanisms that underlie the technical application of a physiological molecule

The potential of antioxidants as tools for lowering the incidence of diseases, including cardiovascular events, is of growing interest. Some antioxidants (e.g. vitamin E and acetyl-salicylic acid) have been described as effective on cardiovascular diseases with mechanisms that differ from other scavenging agents. Currently, vitamin C is used to open occluded long-term central venous catheters, which avoids the process of reinserting a new one and injuring the patient. In this work, we investigated the vitamin C antihemostatic profile by evaluating its effects on the coagulation process. We used different assays, including prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and ancrod time tests. We also examined the overall pH disturbance caused by vitamin C at different concentrations and its effect on the thrombin-initiated fibrin polymerization assay. Our results revealed a significant anticoagulant activity of vitamin C at high plasma concentrations (surpassing the normal 100 ?mol/L ratio) in a cell-independent mechanism. Our results suggest that vitamin C may affect blood coagulation by a direct impairment of fibrin assembly and further formation of a cohesive clot microstructure. This study supports the literature that points to the antihemostatic ability of antioxidant agents, and clarifies the mechanism of vitamin C in opening occluded long-term central venous catheters.

Cold-Stored Platelets in PAS Exhibit Superior Hemostatic Potential

Background: Platelet transfusion is vital for hemostasis in patients with severe bleeding. Currently, platelets are stored at room temperature (RT) for 5 days with gentle agitation. However, this method results in a platelet storage lesion characterized by loss of platelet function and elevated risk of bacterial contamination. It has been shown that refrigeration of platelets at 4o C mitigates adverse effects and other logistical difficulties. Recently, FDA approved the use of cold-stored apheresis platelets for the resuscitation of actively bleeding patients for up to 3 days (5. 21 CFR 606.65(e) & 610.53(c)). We hypothesized that the refrigeration of platelets stored in 65% platelet additive solution (PAS), would better preserve the platelet contractile apparatus and overall hemostatic function. We measured clot stiffness, strength, crosslinking density and platelet contractile forces (PCF) using cone and plate rheometry; and quantified the microstructural architecture of the clots. To corroborate the rheological and morphological measurements with the clotting mechanism, we assayed thrombin generation, FXIII activity, clot retraction, and cytoskeletal rearrangement.Methods: Apheresis platelet (AP) concentrates collected from 4 healthy donors were isolated and stored in 65% Platelet additive solution (PAS) for 5 days at RT (22-24°C) or 4C (4°C). Clotting function was assayed after re-suspension in fresh frozen plasma (FFP). After initiating clotting with 20mM Ca2+, 100µL of the PRP sample was placed on the rheometer plate and clot stiffness was measured for 30 mins at 37o C at a small strain of 1Hz by tracking the evolving storage modulus (G'). Normal force measurements were made to quantify the force generated in the samples as a result of platelet contractile forces. Subsequently, a progressively increasing strain of 1 to 250% was applied until the clot was irreversibly deformed to estimate the yield point (clot strength). Scanning Electron Microscopy was performed on the clots from the rheological analysis to study the clot microstructure. Thrombin generation and FXIII activity were determined using Calibrated Automated Thrombogram and activity assays, respectively. Further, the cytoskeletal rearrangement was studied by activating the washed platelets with thrombin/ TRAP for 5, 10 and 15 seconds and estimating the G-actin to F-actin turnover by Western Blots. The results were analyzed using one-way ANOVA and a post hoc Tukey test for multiple comparisons. The groups were considered statistically significant for p values less than 0.05.Results: The rheological studies on clotting indicate that cold stored platelets form clots that have comparable properties to fresh platelets. RT-stored platelets formed weaker clots (Fig. 1A, B). These findings supported by the evidence of increased FXIII cross-linking activity (Fig.1C). Further, the normal force measurements indicate reduced force generation in RT-stored platelets which imply decreased platelet contractility (Fig. 1D). We also observed differences in geometry and orientation of individual fibrin fibers between fresh and stored platelets. Our preliminary results suggest that thrombin generation potential, crosslinking density, and platelet contractility may contribute to the mechanical strength and morphological differences between fresh and platelets stored at cold and room temperature.Conclusion: Cold-stored platelets form structurally and mechanically stable clots compared to 5 day stored RT platelets as demonstrated by the rheological and microstructural studies. Together, these data suggest that 4C platelets are hemostatically more active than the current standard-of-care and may offer a better solution to patients for resuscitation of bleeding trauma patients.Figure 1 Mechanical testing of evolving clot by rheometry. (A) RT-stored platelets form clots with decreased clot stiffness (G') compared to fresh (F) and cold-stored (4C) platelets (B) Clot strength was defined as the yield point where the clot deformed irreversibly. Clots formed from RT-stored platelets offer less resistance to strain; (C) Relative Cross Linking Density was estimated as the slope of stress-strain curve obtained from amplitude sweep. RT-stored platelet clots show less crosslinking; (D) less contractile force generation in RT platelets. * p <0.05, n=4. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels.

Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

Fractal dimension: A novel clot microstructure biomarker use in ST elevation myocardial infarction patients

ObjectivesChanges in clot microstructure are increasingly implicated in the pathology of atherosclerosis although most data are from techniques in the remote laboratory using altered blood. We validate the novel biomarker Gel Point in STEMI patients and assess therapeutic interventions. Gel Point marks the transition of blood from a visco-elastic liquid to visco-elastic solid and is rapidly measured using unadulterated blood. The Gel Point provides measurements of three parameters to reflect clot microstructure (fractal dimension (df)), real-time clot formation time (TGP) and blood clot strength (elasticity at the Gel Point (G′GP)). MethodsWe prospectively recruited 38 consecutive patients with STEMI undergoing primary percutaneous coronary intervention (pPCI). Venous blood samples were collected on admission, after pPCI and 24 h after admission for assessment of the new biomarkers, standard coagulation tests and scanning electron microscopy (SEM). Resultsdf after pPCI was lower than df on admission (mean 1.631 [SD 0.063] vs 1.751 [0.052], p < 0.000001) whereas df at 24 h was similar to that on admission. G′GP also showed similar trend to df (p < 0.001). TGP was prolonged at after-PCI measurement compared with admission (median 854 s [IQR 581–1801] vs 217 [179–305], p < 0.00001). Changes in the values of df and G′GP were consistent with changes in the SEM images of the mature clot. ConclusionsWe characterise Gel Point derived markers of clot microstructure in patients admitted with emergency arterial thrombosis. This point of care test can potentially be used to assess the efficacy of therapeutic interventions by measuring changes in clot microstructure.

The changes in clot microstructure in patients with ischaemic stroke and the effects of therapeutic intervention: a prospective observational study.

BackgroundStroke is the second largest cause of death worldwide. Hypercoagulability is a key feature in ischaemic stroke due to the development of an abnormally dense clot structure but techniques assessing the mechanics and quality of clot microstructure have limited clinical use. We have previously validated a new haemorheological technique using three parameters to reflect clot microstructure (Fractal Dimension (df)) ex-vivo, real-time clot formation time (TGP) and blood clot strength (elasticity at the gel point (G’GP)). We aimed to evaluate these novel clotting biomarkers in ischaemic stroke and changes of clot structure following therapeutic intervention.MethodsIn a prospective cohort study clot microstructure was compared in ischaemic stroke patients and a control group of healthy volunteers. Further assessment took place at 2–4 hours and at 24 hours after therapeutic intervention in the stroke group to assess the effects of thrombolysis and anti-platelet therapy.Results75 patients (mean age 72.8 years [SD 13.1]; 47 male, 28 female) with ischaemic stroke were recruited. Of the 75 patients, 32 were thrombolysed with t-PA and 43 were loaded with 300 mg aspirin. The following parameters were significantly different between patients with stroke and the 74 healthy subjects: df (1.760 ± .053 versus 1.735 ± 0.048, p = 0.003), TGP (208 ± 67 versus 231 ± 75, p = 0.05), G’GP (0.056 ± 0.017 versus 0.045 ± 0.014, p < 0.0001) and fibrinogen (3.7 ± 0.8 versus 3.2 ± 0.5, p < 0.00001). There was a significant decrease in df (p = 0.02), G’GP (p = 0.01) and fibrinogen (p = 0.01) following the administration of aspirin and for df (p = 0.003) and fibrinogen (p < 0.001) following thrombolysis as compared to baseline values.ConclusionPatients with ischaemic stroke have denser and stronger clot structure as detected by df and G’GP. The effect of thrombolysis on clot microstructure (df) was more prominent than antiplatelet therapy. Further work is needed to assess the clinical and therapeutic implications of these novel biomarkers.

A new structural biomarker that quantifies and predicts changes in clot strength and quality in a model of progressive haemodilution

IntroductionWe investigated the effect of progressive haemodilution on the dynamics of fibrin clot formation and clot microstructure using a novel rheological method. The technique measures clotting time (TGP), clot strength (G`GP), and quantifies clot microstructure (df) at the incipient stages of fibrin formation. We use computational modelling to examine the relationship between structure and mass, as well as helium ion microscopy (HIM) to compare morphological changes in the fully formed clot to that of the incipient clot. MethodsThis is an in vitro study; 90 healthy volunteers were recruited with informed consent and a 20ml sample of whole blood obtained from each volunteer. Five clinically relevant dilutions were investigated using 0.9w.v isotonic saline (0, 10, 20, 40 and 60%, n=18 for each dilution). The rheological method of assessing structural clot changes was compared against conventional coagulation screen and fibrinogen estimation. ResultsFractal dimension (df) and final clot microstructure both decreased with progressive dilution (significant at a dilution of 20%) with similar relationships observed for final clot characteristics in HIM images. Significant correlations were observed between df and G`GP (clot strength) (0.345, p=0.02), as well as clotting time (PT: −0.690, p>0.001; APTT: −0.672, p>0.001; TGP: −0.385, p=0.006). ConclusionsThis study provides new insight into the effects of haemodilution by isotonic saline on clotting time (TGP), clot strength (G′GP) and clot microstructure (df). Previous studies have attempted to link clot microstructure to clot quality/strength, however this study provides a significant step in quantifying these relationships.

Validation of fractal dimension of the incipient blood clot in ST-segment-elevation myocardial infarction

BackgroundMore than 200 combinations of antiplatelet and anticoagulant drugs can be given during an acute cardiac event. However, no biomarker is available to assess their global effect on clot formation and structure. Fractal dimension (Df) and clot formation time (TGP) are global markers of haemostasis that have been validated in healthy and anticoagulated blood. In contrast to standard coagulation assays, these biomarkers use unadulterated whole blood and are measured immediately at the bedside. Df quantifies clot microstructure whereas TGP is a real-time measure of clotting time, and both are calculated from the gel point (GP). We aimed to validate Df and TGP in ST-segment-elevation myocardial infarction (STEMI) and assess the effect of therapeutic intervention. MethodsWe recruited prospectively patients with STEMI undergoing primary percutaneous coronary intervention (PCI). Venous blood samples were collected on admission (after 300 mg of aspirin), after PCI (after clopidogrel, heparin, and bivalirudin), and 24 h after admission. In addition to assessment of Df and TGP, each sample was assessed by standard coagulation tests and a full blood count. Wilcoxon signed-rank and paired t tests were used to compare changes from admission to after PCI and at 24 h. Spearman's correlation analysis was done to explore any associations. Normality was assessed with the Shapiro-Wilks test. This study was approved by the local Research Ethics Committee (REC Number 07/WMW02/34) and written informed consent was obtained from all participants. Findings33 patients (mean age 69 years [SD 12]; 19 men, 14 women) were included. Df on admission (mean 1·75, SD 0·05) was higher than values we have previously reported in healthy individuals despite use of antiplatelet therapy. Df after PCI was significantly lower than Df on admission (mean 1·64 [SD 0·06] vs 1·75 [0·05], p<0·0001) whereas Df at 24 h was similar to that on admission. TGP was significantly prolonged at after-PCI measurement compared with admission (median 723 s [IQR 492–1793] vs 210 [173–314], p<0·0001). Df correlated negatively with TGP (r=−0·75, p<0·0001), prothrombin time (r=−0·49, p<0·001), and partial thromboplastin time (r=−0·52, p<0·0001) but positively with fibrinogen (r=0·36, p=0·0009). There was no significant correlation observed between Df and platelets or haematocrit. InterpretationTherapeutic manipulation in STEMI especially with drugs given during PCI had a striking effect on clot structure and rate of coagulation as measured by Df and TGP. Larger studies are in progress to evaluate potential therapeutic strategies based on these biomarkers and their accuracy in detecting hypocoagulable and hypercoagulable states, and predicting cardiovascular risk. FundingNational Institute for Social Care and Health Research, Engineering and Physical Sciences Research Council.

The pathophysiology, diagnosis and treatment of the acute coagulopathy of trauma and shock: a literature review.

The acute coagulopathy of trauma and shock is associated with significant mortality and, currently, there are no validated laboratory tests which allow for a rapid recognition and treatment of this condition. Therefore, early detection of any clot abnormality in trauma could improve the diagnosis of trauma-associated coagulopathy and subsequent interventions. Review of the literature. The standard laboratory tests, including prothrombin time and activated partial thromboplastin time, are unreliable and describe only an isolated fragment of the complex coagulation pathways. Additionally, thromboelastography and thromboelastometry operate in a non-linear regime which implies that clot formation is the product of both the clotting process and the effect of the measurement. The assessment of the clot microstructure using a scanning electron microscope has resulted in a subjective analysis of a clot structure, showing also poor correlation between the coagulation pathways and clot development. The fractal dimension provides information on the structure and quality of the initial clot, which subsequently acts as a template for how the mature clot will behave. However, these data require further verification in an in vivo setting. At present, the treatment of the coagulopathy is delivered by empirically administered massive transfusion protocols, which lack a specific target for replacement therapy. There is enough evidence to demonstrate that we urgently need a robust test, which would determine and quantify both the rate and the extent of coagulation abnormalities. This could help to tailor the treatment of coagulopathy according to the patient's needs.

P256 Treatment of obstructive sleep apnoea syndrome with continuous positive airways pressure alters haemostasis: further data on the use of fractal analysis to measure microstructure of incipient clot

IntroductionUntreated obstructive sleep apnoea syndrome (OSAS) increases cardiovascular risk and altered haemostasis is at least partly implicated. As previously reported using fractal analysis and a new biomarker called fractal dimension...

Fractal discrimination of random fractal aggregates and its application in biomarker analysis for blood coagulation

A recent rheological study has established that the fractal dimension, df, of an incipient clot, formed at the Gel Point (sol–gel transition) of coagulating blood is a significant new biomarker of haemostasis. In whole healthy blood, incipient clots show a clearly defined value of df=1.7 within a narrow range, which represents a new ‘healthy index’ for normal clotting. The addition of unfractionated heparin significantly prolongs the onset of clot formation with a corresponding reduction of df as a function of heparin dose. However, as clots mature they exhibit (i) an expected increase in df and (ii) a significant increase to spread of these values, i.e. df’s in the range 2.0–2.5, limiting the use of df as a discriminant of clot microstructure.The present study, details how and why the spectral dimension, ds, can be used to accommodate this shortcoming and allow discrimination of mature forms of clot microstructure in indistinguishable in terms of their fractal dimension. To elucidate why ds permits discrimination a numerical experiment was conducted on computationally generated random fractal aggregates (RFAs) with a priori set value of df. Starting from RFAs with a df of 1.7, mature RFAs are evolved from these incipient templates by two differing growth processes achieving a final df of 2.1. Fractal and statistical analysis of the mature RFAs reveals, for the first time, that their differing internal structure is manifest in the magnitude of ds. The potential clinical significance of these findings is discussed in terms of the possibility of exploiting the incipient clot’s ability to template the internal arrangement of the mature clot to better predict long term clot susceptibility to lysis.

Rheometrical detection of incipient blood clot formation by Fourier transform mechanical spectroscopy

We report a study of the coagulation of whole blood by oscillatory shear Fourier transform mechanical spectroscopy. The results include the first identification of the Gel Point of coagulating blood in terms of the Chambon–Winter Gel Point criterion and we show how this may be used as an appropriate basis for detecting the establishment of an incipient clot in samples of human blood. A comparison of this technique with results obtained from a thromboelastograph (TEG) indicate that the latter is not capable of detecting the incipient clot, whose establishment can occur several minutes prior to the first recorded TEG response. Our results also reveal, for the first time, the incipient clot's fractal characteristics. An analysis of the viscoelastic data obtained at the Gel Point of samples of whole blood drawn from healthy individuals indicate that the incipient clot is characterised by a fractal dimension d f = 1.74(±0.07). The significance of these findings is discussed in terms of their potential application in studies of pathologically induced changes in blood clot microstructure.

Confocal microscopy movies of fibrin clots during ultrasound-accelerated thrombolysis

Blood clots made of human purified fibrin (white clots) were insonified with 1 MHz pulsed ultrasound during observation by fluorescence confocal microscopy. A deconvolution microscope allowed extremely thin sheets (0.2 μm) of fibrin to be viewed at a resolution of 0.2 μm per pixel, and the clot microstructure visualized. Acoustic pressure amplitudes from 0.1 to 0.8 MPa (peak-to-peak) were inferred using the image blur of 0.6-μm-diameter polystyrene spheres coated with FITC fluorecent label present in the clots. Acoustic pulse widths of 1 ms and pulse repetition frequencies of 125 Hz reduced clot heating to less than 3°C during each 30-minute exposure. Still 100 μm by 100 μm images were recorded every 10 seconds during pauses in insonificaiton, to produce time-lapse movies that are compared with movies made during sham ultrasound exposures.

Rennin Modification of Bovine Casein to Simulate Human Casein Composition: Effect on Acid Clotting and Hydrolysis by Pepsin.

Human milk formed fine, dispersible floes at pH 4, in contrast to large clots from bovine milk. In order to simulate human casein composition (primarily β-casein), bovine casein (2%, pH 7, 11mM CaCl2) was selectively coagulated with rennin (0.1–0.5mg/g casein) and heating. Electrophoretic analyses indicated an increase in β-:αsl-casein ratio from 0.7 to 3, with protein recoveries of 20–30%. β-Casein rich fractions were obtained from casein or skimmilk modified at 37°C or 4°C, although partial hydrolysis of β-casein occurred at 4°C. At pH 2, pepsin digestibility of the clear or turbid solutions followed the order αsl-casein > β-casein, and bovine control > modified caseins or milks. This trend was reversed at pH 4, the small loose clots of β-casein and modified samples being hydrolyzed more readily than the large hard clots of αsl-casein and bovine control samples. Human casein and milk showed low digestibility at pH 2 and 4, and were shown by electron microscopy to consist of tightly clustered clots. Thus, both casein composition and clot microstructure affect pepsin digestibility. The loose clot and high digestibility at pH 4 of rennin-modified samples may be advantageous for infant feeding.