Interpretation and Validation of Maximum Absorbance Data Obtained from Turbidimetry Analysis of Plasma Clots

Abstract

Turbidimetry is used to characterize fibrin clot properties. In purified systems, maximum absorbance (MA) directly relates to fibrin fiber cross-sectional area. However, in plasma samples there are discrepancies in the relationships between MA and fibrinogen concentration, fiber diameter, other clot properties, and cardiovascular disease outcomes, which complicate data interpretation. This study aims to advance understanding of MA of plasma clots through testing how well it relates to fundamental dependence on fibrinogen concentration and fiber diameter as predicted by light scattering theory, other clot properties and lifestyle, and biochemical variables. Plasma samples from 30 apparently healthy individuals with a fibrinogen concentration from 2.4 to 6.4 g/L were included. We performed turbidimetry, permeability, scanning electron microscopy, and rheometry on in vitro formed plasma clots. MA correlated more strongly with fibrinogen concentration (r = 0.65; p < 0.001) than with fiber diameter (r = 0.47; p = 0.01), which combined explained only 46% of the MA variance. Of additional variables measured, only low-density lipoprotein cholesterol correlated with MA (r = 0.46; p = 0.01) and clot lysis (r = 0.62; p < 0.0001) but not with fiber diameter or fibrinogen concentration. MA correlated with clot lysis time (r = 0.59; p = 0.001), storage modulus (r = 0.61; p = 0.001), and loss modulus (r = 0.59; p = 0.001), and negatively with clot permeability (r = –0.60; p = 0.001) also after adjustment for fibrinogen concentration and fiber diameter. Increased MA is indicative of a prothrombotic clot phenotype irrespective of fibrinogen concentration. MA is more indicative of overall clot density than of fiber diameter. Other plasma components can alter internal fiber density without altering fiber diameter and should be considered when interpreting MA of plasma samples.

Effects of Diabetes Mellitus on Fibrin Clot Structure and Mechanics in a Model of Acute Neutrophil Extracellular Traps (NETs) Formation

“…Furthermore, our results confirm previous studies by showing a positive correlation between the stiffness of clots and fibrinogen concentration [ 20 , 25 , 26 ]. In addition, higher fibrinogen levels were significantly associated with higher levels of onset stress for strain-stiffening, while the corresponding onset strain was negatively associated with fibrinogen levels.…”

“…Our finding shows that this decreased rate of fibrinolysis in clots prepared from subjects with DM is also found in a model of acute NETs formation and independent of fibrinogen level. In addition, we found a significant negative correlation between the rate of fibrinolysis and fibrinogen concentration and clot stiffness, confirming previous data [ 20 ].…”

“…Significant positive correlations between fibrinogen, glucose or HbA1c levels and fiber diameter were found, corresponding to previous reports [ 5 , 20 ]. This shows that worsened glycaemic control, as shown by increased HbA1c levels, is associated with thicker fibers [ 5 ].…”

“…In our study, we observed a significantly higher concentration of fibrinogen in subjects with DM (3.8 ± 0.7 g/L) compared to subjects without DM (3.3 ± 0.6 g/L) ( p = 0.01). Since fibrinogen concentration is known to affect clot properties [ 20 ], we first determined the correlation of the fibrinogen concentration with the clot parameters in a model of acute NETs formation. Fibrinogen levels were positively associated with fiber diameter (Spearman’s correlation coefficient (r s ) = 0.60, p = 0.01) ( Table 2 ).…”

Reductions in plasmin inhibitor and fibrinogen predict the improved fibrin clot lysis 6 months after obesity surgery

“…Our results also demonstrate that lowering of cholesterol after RYGB independently predicts the reduction in PI. No other study has looked at the association between cholesterol and the inhibitory capacity of PI, but a prolonged clot lysis time was previously associated with low HDL‐cholesterol 37 and high LDL‐cholesterol, 38 and Pieters et al suggested that LDL‐cholesterol can bind to fibrin clots thereby hindering the action of lytic enzymes, a new role that deserves further investigation 38 …”