Measurement of functional and immunologic levels of tissue factor pathway inhibitor. Some methodologic considerations

Tissue factor pathway inhibitor (TFPI) is a newly identified inhibitor of proteases generated during activation processes. Several functional methods based on a chromogenic substrate technique measuring residual tissue thromboplastin/FVIIa catalytic activity using excess factor X and a chromogenic substrate for FXa have been published. Recently, a sandwich ELISA method has become available. A modified functional method was compared with the ELISA based antigen method in several groups of normal individuals. The functional assay is sensitive to TFPI concentrations of 125-200 ng/ml based on a rTFPI standard. The immunologic method is sensitive from 0 to 400 ng/ml. A marked dichotomy was observed between the two methods. The ratio between TFPI antigen and functional levels varies widely in patient groups. This TFPI standard was supplemented with buffer and normal human plasma and functional TFPI was measured before and after heat treatment at 56 degrees C for 10 min. Heat treatment of plasma (buffer) after the addition of TFPI resulted in decreased functional TFPI compared with the addition of TFPI after the plasma (buffer) was heat treated. In contrast, the immunologic method does not require heat treatment. Addition of exogenous TFPI to plasmas obtained from normal and the above patient plasmas demonstrated that recovery of functional TFPI activity was plasma dependent. Plasma proteins markedly influenced functional and antigenic levels of TFPI. The TFPI standard added to human pooled plasma gave varying recoveries in the immunologic and functional assays. These results indicate that currently available methods may provide highly variable results on TFPI levels. Several matrix related effects should be taken into account for proper evaluation of TFPI.