FLOW CYTOMETRIC ANALYSIS OF RETICULATED PLATELETS - EVIDENCE FOR A LARGE PROPORTION OF NONSPECIFIC LABELING OF DENSE GRANULES BY FLUORESCENT DYES

The labelling of platelets with thiazole orange (TO) has been utilized by various laboratories to determine the percentage of reticulated pl atelets within whole blood or platelet-rich plasma (PRP). A proportion of TO labelling, however, is not entirely mRNA specific and remains t o be fully defined. Almost half of the total TO-positive signal within normal platelets (n = 5) was shown to be abrogated upon degranulation with 80 mu M thrombin receptor activating peptide (TRAP) (P=0.006), s trongly suggesting that platelet granules are non-specifically labelli ng with dye. We have confirmed this hypothesis by studying TO labellin g of platelets within whole blood from dense granule deficient patient s, e.g. Hermansky-Pudlak syndrome (HPS) (n=5) and storage pool disease (SPD) (n=4). The levels of TO-positive platelets were found to be sig nificantly lower than normal (P=0.0003 and P=0.0002 respectively), but not significantly different from TRAP degranulated platelets. Upon de granulation of HPS and SPD platelets there was very little further red uction in the TO signal. Incubation of normals and SPD whole blood wit h different concentrations of either TO or coriphosphine-O confirmed t hat dense granules were non-specifically labelling even at high concen trations of both dyes. These findings suggest that although TO labelli ng is in part RNA specific, the dense granular pool of nucleotides app ears to cause a substantial amount (similar to 50%) of non-specific la belling observed under these conditions of assay. This can easily be c ontrolled for by a degranulation step with a non-enzymatic platelet ag onist such as TRAP, and may have important consequences for the eventu al standardization, clinical utilization and automation of reticulated platelet assays.