Thromboelastography in the assessment of coagulation changes in peripheral vascular disease

Studies up to date in the western literature involving changes in coagulation in patients with peripheral vascular disease(PVD) have consistently reported activation of coagulation based on measurement of raised levels of markers of activation of coagulation(clot formation), decreased fibrinolysis (breakdown of fibrin), platelet activation leading to increased aggregation and defective endothelial (arterial wall) function in patients with PVD that leads to thrombosis in arterial circulation thus designating this group of patients as hypercoagulable.‘ Interestingly these patients are not deemed as such in clinical practice as the routine coagulation tests(RCTs) currently employed do not identify these abnormalities. The overall purpose of the research described in this thesis was to identify changes in coagulation in patients with peripheral vascular disease using Thromboelastography (TEG), a technique which exploits the visco-elastic properties of a blood clot as it forms, retracts or lyses (breaks down) using a small volume of whole blood. Validation of TEG technique employing citrated whole blood (CWB) and heparinase modified (hepTEG) methods and standardisation of analysis to overcome variability in TEG analysis, blood sample instability due to storage times was carried out. TEG analysis after sample storage between 1-2 hours showed no significant inter or intra sample assay variability confirming excellent reproducibility( (p= NS; reliability coefficients (Alpha) greater than 0.9 for R time, K time, Angle, Maximal Amplitude (MA) and Coagulation Index(CI)). hepTEG method confirmed complete reversal of heparin effect in blood samples obtained from participants following heparin administration.(R ii time: 9.16+/- 1.68 Vs 8.90 +/- 1.48 p=NS; K time: 2.98+/- 0.87 Vs 3.20 +/- 1.05 p=NS; Angle: 56.86+/- 6.39 Vs 56.90 +/- 5.30 p=NS; MA 56.34+/- 6.39 Vs 56.79+/-6.23 p=NS; CI: 0.59+/-1.19 Vs 0.88+/-1.00 p=NS). 50 age matched controls to obtain reference values for comparison and documentation of any significant changes in TEG parameters due to age, gender anaesthesia and surgery was completed. Preparation /induction of anaesthesia led to a significant trend towards activation of coagulation in all age groups and gender (Angle 56.23 +/-0.89, CI 54.42, 58.04 Vs 58.21+/- 0.83, CI 56.54, 59.88;p=0.003 and MA 56.40 +/- 0.79 CI 54.80, 58.01 Vs 59.36 +/- 0.89, CI 57.56, 61.15; p=0.003 and CI 0.71 +/- 0.14, CI 0.42, 1.00 Vs 1.35 +/- 0.17, CI 1.00, 1.69;p=0.003) Surgical stimulus led to a further activation of coagulation that followed induction of anaesthesia (Angle: 58.21+/- 0.83, CI 56.54, 59.88 Vs 60.98 +/- 0.69, CI 59.59, 62.37; p=0.001). TEG parameter values obtained from healthy controls aged over 45 are used as reference values for this study. TEG showed no significant differences in TEG parameters when samples obtained from an upper limb artery and a vein were analysed. However when samples from the main lower limb artery (common femoral artery/CFA) and the major vein that drains the same limb (common femoral vein/CFV) in patients with symptomatic PVD, TEG identified significant activation of coagulation in samples obtained from the vein that drains an ischaemic limb (decrease in R time (p0.05), an increase in MA (p0.05) and an increase in CI (p0.002)). This interesting finding led to a hypothesis that ischaemic tissue has a prominent role in the activation of coagulation observed in iii patients with PVD. To test this hypothesis, common femoral venous and arterial samples from 30 patients with symptomatic PVD were analysed using TEG. TEG identified significant activation of coagulation in samples obtained from an artery downstream (CFA) when compared to those obtained from an artery that is proximal (Aorta). This change towards hypercoagulation was also found to be positively related to the degree of narrowing or stenotic disease (quantified using angiography) between these two sampling points(R, r=0.442, p0.05 / MA, r=0.379, p0.05 / CI r=0.429, p0.05). A significant positive relationship in between degree of ischaemia (ABPI) and difference in TEG parameter values in between arterial and venous blood samples obtained from an ischaemic limb (ABPI) on that side (CI v ABPI r = -0.427 p0.05, MA v ABPI r =-0.370 p0.05) was also found in this study. These findings suggest activation of coagulation as the blood flows down an atherosclerotic vessel and in combination with the observed changes due to the presence of peripheral ischemia tissue suggested that the hypercoagulability observed in PVD may have its origins in the ishaemic limb itself. To clarify the role of non-ionic contrast media (NICM) in the context of conflicting findings regarding its thrombogenic potential especially in patients with PVD undergoing angiography, aortic blood samples(n=30) were obtained before and after injection of NICM. Heparinase modified TEG analysis showed that there was no activation of coagulation immediately after NICM exposure and in fact there was a significant trend towards hypocoagulation in contrast to the published reports of increased iv thrombogenicity after NICM exposure (increase in R time (time to fibrin formation) (CI 7.8,10.18 minutes) (p=0.036), in K time (dynamics of clot formation) (CI 2.2,2.8 minutes) (p=0.028), and a reduction in Angle (decreased acceleration of fibrin build up) (CI 53.10,62.7 degrees) (p=0.013),MA (reduced ultimate clot strength) (CI 54.5,62.7 mm) (p=0.013) and (CI) (decreased overall coagulation status) (0.31,1.95) (p=0.032)). This study also showed that despite this significant reduction in the activation of coagulation after NICM exposure, PVD patients were consistently procoagulant when compared to age- matched controls(n=30) who were not exposed to NICM (R time: p=0.029/K time: p=0.001/Angle: p=0.003/MA: p=0.020 and CI: p=0.014) Patients with ischaemic heart disease, a consequence of significant coronary artery atherosclerosis have reduced amounts of naturally occurring anticoagulant substances like heparan, heparan-sulphate proteoglycan and endogenous heparin. Since patients with PVD exhibit similar pathology in addition to the presence of peripheral ischaemic tissue, to identify any similar defect in PVD, blood samples from patients with symptomatic PVD(n=28) and age matched control subjects were analysed using hepTEG. Heparinase modified TEG analysis identified for the first time, heparinase sensitive heparin-like activity in peripheral venous samples in patients with PVD (R time 7.50 0.44 min / CI 6.54, 8.46 Vs 7.17 0.40 min / CI 6.30, 8.05 / p=0.041). Endogenous heparin-like activity is found to be reduced in PVD and this reduction also correlated with the degree of peripheral ischaemia v(ABPI) (correlation coefficient: abpi: 1.000 / change in R time 0.350 / p=0.021)TEG analysis of samples from 30 patients with aortic aneurysmal disease and 14 aortic occlusive disease undergoing revascularisation and 30 controls were carried out using hepTEG method. Baseline TEG parameter values in the aneurysm group showed significant activation of coagulation when compared to the controls (R time (p=0.001); K time (p=0.008) and (CI) (p=0.047)). Following release of the aortic cross-clamp a significant trend towards activation of coagulation was noted in the aneurysm group and a similar but pronounced activation of coagulation was noted in the occlusive group, confirming activation of coagulation following reperfusion of ischaemic tissues (R time (p=0.042); K time (p=0.043) MA (p= 0.034) and CI (p=0.026)). Further TEG analysis during postoperative period showed that both these patient groups exhibit sustained periods of hypercoagulability. In addition to these findings hepTEG method further revealed underlying hypercoagulability, despite exogenous heparin administration, confirming presence and activation of non- AT-III dependent pathways of activation of coagulation in these patients. TEG analysis of blood samples from 86 patients admitted with symptoms peripheral ischaemia requiring revascularisation surgery or amputation were carried out and TEG parameter values obtained from control group was used for compari