Detection and quantification of circulating immature platelets: agreement between flow cytometric and automated detection

Abstract

Immature platelets-also termed reticulated platelets (RP)-are platelets newly released into the circulation, and have been associated with a variety of pathological thrombotic events. They can be assessed by flow cytometry after staining with thiazole orange (TO) or by using a module added to a fully automated analyzer that is currently in wide clinical use and expressed as a fraction of the total platelet count (IPF). We sought to assess the correlation and agreement between these two methods. IPF was measured using Sysmex XE 2100-and at the same time point- we used TO staining and flow cytometry to measure RP levels. Two different gates were used for the flow cytometry method, 1 and 0.5 %. Measurements from the automated analyzer were then compared separately to measurements performed using each gate. Agreement between methods was assessed using Bland-Altman method. Pearson's correlation coefficient was also calculated. 129 subjects were enrolled and stratified into 5 groups: (1) Healthy subjects, (2) End stage renal disease, (3) Chronic stable coronary artery disease, (4) Post Coronary artery bypass surgery, (5) Peripheral thrombocytopenia. Median IPF levels were increased for patients in groups 2, 3, 4 and 5 (4.0, 4.7, 4.3, and 8.3 % respectively) compared to healthy subjects (2.5 %) p = 0.0001. Although the observed correlation between the two methods tended to be good in patients with high IPF values (i.e., group 5), the overall observed correlation was poor (Pearson's correlation coefficient r = 0.27). Furthermore, there was poor agreement between the two methods in all groups. Despite the good correlation that was observed between the two methods at higher IPF values, the lack of agreement was significant.

Evaluation of the immature platelet fraction contribute to the differential diagnosis of hereditary, immune and other acquired thrombocytopenias

“…13 or using automated hematological analyzers, as in the study by Myiazaki 14 and ours. Few studies have formally evaluated the correlation of IPF measurements by these two methods but in two of the most recent ones, this correlation was limited to ITP patients 11, 30 . Considering the challenges of standardizing platelet flow cytometry analyses compared to a CBC, we believe that it is fair to state that the former method should not be regarded as a gold-standard for IPF measurement.…”

Platelet Acetyl-CoA Carboxylase Phosphorylation

“…First, phosphoACC was determined using immunoblotting in whole platelet lysates. Given the circulating platelets’ heterogeneity (32), it is still unclear whether increased phosphoACC occurs in the entire platelet population or in a subgroup only. Moreover, in this setting, platelets must be purified before phosphoACC is measured, and quantitative assessment of phosphoACC remains challenging.…”

It's reticulated: the liver at the heart of atherosclerosis

“…Although the terms 'reticulated platelets' and 'immature platelets' are used interchangeably, reticulated platelets are detected by flow cytometry with thiazole orange staining of the high RNA content in a reticulated pattern (hence the name 'reticulated platelets') (Kienast & Schmitz 1990), while 'immature' platelets are detected on automated cell counters with an add-on module allowing platelets to be stained with a polyamine/oxazine combination (to detect RNA). Therefore, it should be noted that reticulated platelets and immature platelets do not represent necessarily identical populations even though there is a considerable overlap (Ibrahim et al 2016).…”