A new approach to personalized assessment of functional platelet disorders in children with inherited thrombocytopenias by flow cytometry.

Abstract

Inherited thrombocytopenias are variable in the count and size of platelets, which is related with the number of receptors and intracellular structure. So, the reference ranges do not allow interpretation of functional disorders, especially in macrothrombocytopenias and microthrombocytopenias. The flow cytometry diagnostic approach to use the reference values is necessary. Seventy-five pediatric patients were divided into three groups by platelet size: 25 with normothrombocytopenia ( RUNX1, ANKRD26, ETV6 , and CYCS ), 25 with microthrombocytopenia ( WAS ), 25 with macrothrombocytopenia ( MYH9, TUBB1, SLFN14 , and BSS ). Platelet size, granularity, GPIb/V/IX, GPIIb/IIIa, granules, and procoagulant platelets were analyzed at rest and after activation by a mixture of TRAP+CRP. In addition to the absolute value indicators, a calculated 'index at rest/after activation' in relative units was introduced for personalized changes assessment. The hemorrhage was assessed using a Pediatric Bleeding Questionnaire. The control consisted of 40 children. The bleeding score ranged from 0 to 20 (median of 2). Upon activation, in all groups, weakening in platelet size contraction and procoagulant platelet formation ( P  ≤ 0.02) were observed. In normothrombocytopenia and microthrombocytopenia groups, increased granularity upon activation, attenuation of CD42b shedding/internalization, correlation ( r  ≥ -0.65) between a decrease of procoagulant platelets less than 5% and increase of bleeding were found. Additionally, reduction of δ-granules ( P  ≤ 0.01) in normothrombocytopenias, attenuation of externalization and activation of GP IIb/III, and granules release ( P  ≤ 0.001) in the microthrombocytopenia were detected. The use of 'activation indexes' in relative units allowed to identify and characterize morphofunctional abnormality patterns in different platelet size thrombocytopenia groups and interpreted control values for detecting personalized patient disorders regardless of platelet size.