Interpretation of maximum absorbance data obtained from turbidimetry in plasma samples with varying fibrinogen concentration
Nunes, Claudia Maria
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Cardiovascular disease (CVD) has become South Africa’s second largest cause of death. CVD is often associated with vascular injury, which initiates the coagulation cascade and formation of an occlusive or non-occlusive thrombus. Clots can be characterised by various properties, which determine their behaviour in the vasculature. Structural clot properties can be determined directly by microscopy and indirectly by turbidimetry. In purified fibrinogen systems, with a fixed fibrinogen concentration, increase in absorbance measurement, obtained from turbidity curves, is used as a proxy marker for fibrin fibre diameter. However, in plasma samples with varying fibrinogen concentrations disagreement exists regarding the interpretation of maximum absorbance, as it also increases with an increase in fibrinogen concentration and may thus not be a true reflection of fibre diameter, but rather of increased clot density. In this study the main aim was to characterise structural clot properties from plasma samples with varying fibrinogen concentrations, using both direct (scanning electron microscopy) and indirect methods (turbidimetry, permeability) to provide clarity on the interpretation of maximum absorbance values from plasma samples. PARICIPANTS AND METHODS Data was collected in the South African Prospective Urban and Rural Epidemiology (PURE) study, in North West, Potchefstroom during 2015 from apparently healthy black women and men (n=900) residing in urban or rural settlements. A sub-sample of 30 participants was systematically selected based on maximum absorbance values and fibrinogen concentrations. The methods pertaining to this study include the following fibrin network determinations: total fibrinogen and fibrinogen Y’ concentration, turbidimetry, permeability, scanning electron microscopy, fibrin content and rheometry. RESULTS In the 30 investigated plasma samples, maximum absorbance showed strong, positive significant correlations with lag time, slope, clot lysis time (CLT), porosity, elastic modulus (G’), ABSTRACT iii viscous modulus (G’’), total fibrinogen concentration and fibre diameter. The correlation between maximum absorbance and total fibrinogen was stronger than between maximum absorbance and fibre diameter. Aside from its significant correlation with maximum absorbance, fibre diameter correlated with slope only. After controlling for total fibrinogen concentration in our samples, we found no significant correlations between fibre diameter and other clot properties, whereas maximum absorbance still significantly correlated with lag time, slope, CLT, porosity, elastic and viscous moduli. Maximum absorbance however no longer correlated significantly with fibre diameter. Three covariates identified for possible contribution to clot property variance, after plasma samples were divided into two groups (low and high maximum absorbance) matched for fibrinogen concentration, were body mass index (BMI), C-reactive protein (CRP) and low-density lipoprotein cholesterol (LDL-C). BMI correlated significantly with CLT, CRP with total fibrinogen concentration, slope, maximum absorbance and porosity and LDL-C with lag time, maximum absorbance and CLT. CONCLUSION From our data obtained from plasma samples with varying fibrinogen concentration, we found both increased maximum absorbance and increased total fibrinogen concentration to be associated with the formation of thicker fibrin fibres. Our results show that although maximum absorbance is related to fibre diameter, in plasma samples with varying fibrinogen concentration, it is not equivalent to fibre diameter. We suggest that maximum absorbance is more likely indicative of clot (protein) density. In addition, other environmental and biological factors including BMI, CRP and LDL-C may influence fibrin clot properties, including maximum absorbance in plasma samples.
- Health Sciences