Tissue factor mRNA quantitation without prior monocyte isolation

Monocyte tissue factor (TF) quantitation evaluates the involvement of coagu lation processes in many diseases. However technical difficulties, such as blood sampling of cells representative of the whole intravascular pool, cel l isolation, protein quantitation or activity assessment, hinder reliable e valuation of TF expression by activated monocytes. Early determination of s uch activation can be achieved through TF mRNA quantitation by RT-PCR and s ensitive product detection, such as automated electrophoresis of fluolescen tly labeled products. Although it is very sensitive, this method has its li mitations. It needs to be standardized using other mRNA that display two ma in characteristics: the absence of up-regulation during inflammation and si milar levels of expression when compared with the target mRNA. Widely used standardization housekeeping genes such as HLA or GAPDH genes only meet the former requirement. We demonstrate here that CD11b gene expression meets b oth conditions. Moreover; because of its specific expression in myelomonocy tic cells, if is possible to avoid further monocyte purification from a reg ular mononuclear cell preparation. A rapid, sensitive, specific and accurat e way to evaluate monocyte TF expression is described in this paper.