Combined accurate platelet enumeration and reticulated platelet determination by flow cytometry.

Background: Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method.

Goal: To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method.

Methods: TO concentration, incubation, and fixation method were determined to be 10% of stock concentration, 30 min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control tube to set the limit of TO staining.

Results: Normal range (n = 51) was 9.9 ± 3.1%. Analysis of 40 patients with immune-thrombocytopenia-purpura (ITP) showed a RP range from 4.3% to 81.2%. Platelet enumeration was consistent with our previous studies in this area.

Conclusions: Combining CD41/CD61 platelet enumeration with TO RP percentage is possible. Accurate RP percentage requires an effective gating strategy, as background fluorescence cursor placement is important. This method for enumeration of RP percentage combined with accurate platelet enumeration, particularly in the low range, should prove useful in differentiating production from consumption issues in thrombocytopenia and monitoring response to therapy.