Measurement of Thrombin-Alpha-2-Macroglobulin Complexes Generated in Plasma

The aim of this study was to evaluate methods to determine the concentration of thrombin-a2-macroglobulin (TAM) complexes in thrombin generation (TGA) reactions. Specifically, methods for standardized and convenient sample transfer from TGA assays to TAM assays were evaluated with the aim to evaluate the impact of TAM in TGA assays and – if necessary – to make the combination of the two assays applicable for clinical routine.

Furthermore, we asked the question whether concentration of TAM complexes during TGA varies significantly in normal samples and in anticoagulated or thrompophilic samples and thus would influence TGA results.

Currently available TGA assays measure not only free thrombin but also a2-Macroglobulin bound thrombin which is measured in such assays and might lead to overestimate the amount of thrombin generated in TGA. Methods which use mathematical correction for this overestimation assume the same concentration of TAM complexes in different types of samples. This is certainly not the case in children, who have much higher concentrations of a2M. For a correct determination of thrombin generated it could be necessary to measure the concentration of TAM complexes in each TGA reaction individually. Recently a new immuno-activity assay for measuring TAM complexes has been developed (Technozym^®TAM ELISA assay).

The TECHNOTHROMBIN^® TGA assay used in this study does not include any fibrin polymerization inhibitors. Thus the sample transfer from the TGA reaction to the TAM assay is hampered by the clot that forms during thrombin generation. For a routine application of both tests, convenient methods to remove the clot without influencing the result of the TAM assay have to be developed.

Thrombin generation was measured using the TECHNOTHROMBIN^® TGA assay.

TAM complexes were measured using TECHNOZYM^® TAM ELISA. The amount of thrombin in TAM complex is expressed in % of the total amount of thrombin generated. Sample material for the TGA assay was citrated plasma. Samples for TAM assay were taken as subsamples after completion of the TGA reaction. The following methods were used for removal of the clot: centrifugation at 13.000 g for 5 min, filtration through 1.2 μm, low protein binding filters using the standardized filtration device CEVERON^® MFU 500. For comparison liquid was just pipetted from the TGA reaction without prior removal of the clot.

In normal plasma samples (pool of 10 donors) the total amount of thrombin generated (= AUC: area under the curve) was 5110 nM ± 321. (Intra assay variation CV< 5%, n=4; inter-assay variation CV<10% N=2). Approximately 1% of total thrombin generated was found in TAM complexes (mean value of TAM/AUC ratio 1,1% ± 0,1%, mean TAM concentration 56,3nM ±3,8). No significant difference in TAM values for clot removal by centrifugation or filtration could be found (n=8; p=0,1). In contrast, pipetting the sample from the TGA reaction without clot removal yielded significantly lower TAM values (n=8; p=0,003). For individual normal samples (n=10) similar mean values were found: 4380 nM ± 281 for AUC, 47± 10 for TAM. The ratio of TAM/AUC was 1,1% ± 0,2.

In heparinized plasma samples (0.1IU/mL Heparin), the percentage of thrombin in TAM was as expected lower than 1% indicating that heparin competes with the formation of TAM complexes.

Anticoagulated samples (n=4) seem to have a tendency to lower TAM/AUC ratios (below 1%) and thrombophilic samples (n=5) above 1% - testing of larger numbers of samples is currently performed to confirm this finding.

The results of the method evaluation show that analyzing of samples from the TGA assay for thrombin-a2-Macroglobulin can be conveniently achieved by removal of the clot by filtration under standardized conditions. This offers possibilities for routine application of the combination of TGA and TAM assays.

Analysis of different samples indicates that TAM complexes might be altered in anticoagulated or thrombophilic state. From this limited study it seems that the amount of TAM in TGA assays does not vary too much in individual groups of samples as TAM would influence TGA results.