Reticulated platelets stained with thiazole orange: methodology and clinical application in the diagnosis of idiopathic autoimmune purpura.

Reticulated platelets (RP) are defined as platelets with residual ribosomal RNA which is related to maturation stage. RP stained with thiazole orange (TO) are a useful marker for bone marrow thrombocytopoiesis and could be a simple test to distinguish thrombocytopenia between autoimmune and aplastic disorders. We evaluated RP of 22 patients (3 males and 19 females), median age 53 years (12-78), affected by idiopathic autoimmune purpura (ITP) at the moment of diagnosis and 61 healthy normal controls. RP were studied utilizing a quantitative fluorescent test on platelet rich plasma (PRP) obtained by centrifugation from peripheral blood. The platelets are fixed with paraformaldehyde, stained with TO and analyzed by flow cytometer. The results were examined by a fluorescence histogram where M1, M2, M3 regions were defined on the basis of the incidence of control platelet populations. The analysis of the results suggests that the number of RP was significantly higher in patients affected by ITP, compared with RP number observed in normal subjects (p<0.001). We obtained consistent TO positive platelet rates in patients with ITP when the platelet count was < 20×109/L compared with to patients with platelet counts > 20×109/L (p< 0.05). Furthermore, the number of RP resulted in a relationship with the platelet count: the presence of M2 and M3 fractions is inversely correlated with the platelet count (p=0.05). We concluded that our method of TO positive RP measuring is helpful for the diagnosis of ITP, especially when bone marrow aspiration is not possible and useful for the evaluation of bone marrow thrombopoiesis in different clinical applications.