Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labelling of dense granules by fluorescent dyes.

Abstract

The labelling of platelets with thiazole orange (TO) has been utilized by various laboratories to determine the percentage of reticulated platelets within whole blood or platelet-rich plasma (PRP). A proportion of TO labelling, however, is not entirely mRNA specific and remains to 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 microM thrombin receptor activating peptide (TRAP) (P = 0.006), strongly suggesting that platelet granules are non-specifically labelling with dye. We have confirmed this hypothesis by studying TO labelling of platelets within whole blood from dense granule deficient patients, 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 significantly lower than normal (P = 0.0003 and P = 0.0002 respectively), but not significantly different from TRAP degranulated platelets. Upon degranulation of HPS and SPD platelets there was very little further reduction in the TO signal. Incubation of normals and SPD whole blood with different concentrations of either TO or coriphosphine-O confirmed that dense granules were non-specifically labelling even at high concentrations of both dyes. These findings suggest that although TO labelling is in part RNA specific, the dense granular pool of nucleotides appears to cause a substantial amount (approximately 50%) of non-specific labelling observed under these conditions of assay. This can easily be controlled for by a degranulation step with a non-enzymatic platelet agonist such as TRAP, and may have important consequences for the eventual standardization. clinical utilization and automation of reticulated platelet assays.