A flow cytometric technique using thiazole orange to detect platelet engraftment following pediatric stem-cell...

Thiazole orange (TO) is a nucleic-acid-specific dye that enters cells without pretreatment. When it binds to either RNA or DNA, there is an increase in fluorescence emission. This property has been utilized to measure the amount of newly released platelets using flow cytometry. These newly released platelets differ from more mature platelets because they still contain residual amounts of RNA, and have become known as reticulated platelets.

Peripheral blood samples were collected at least 48 h following platelet infusion. For validation, manual reticulocyte counts obtained in the laboratory were compared with results obtained using TO and flow cytometry. Following validation, experiments using platelet-rich plasma were performed to evaluate the presence of reticulated platelets in the sample.

Validation experiments comparing the manual and flow cytometric reticulocyte counts gave a strong relationship between the two values (r(2) = 0.92). Reticulated platelet studies performed on platelet-rich plasma samples yielded the following results. Patients who did not engraft within 4 days were significantly different from patients who did engraft within 4 days, idiopathic thrombocytoperic purpura (ITP) patients, and donor platelet segments (all P < 0.0008). Patients who engrafted within 4 days, ITP patients, and donor platelet segments were all statistically similar (all P > 0.08).

The statistical difference between patients who did engraft within 4 days and those who did not suggests that this method could have an important clinical impact in determining those patients who are still in need of platelet support. However, great care must be taken when performing and analyzing the results.