Functional platelet defects in children with severe chronic ITP as tested with 2 novel assays applicable for low platelet counts

Abstract

Key Points• Pediatric chronic ITP patients with a severe bleeding phenotype exhibit functional platelet defects.• The platelet microaggregation test and the platelet reactivity assay are able to assess platelet function at extremely low platelet count.Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 mL of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts ( <10 9 ). These tests show that platelet function is related to bleeding phenotype in chronic ITP. (Blood. 2014;123(10):1556-1563

Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP

“…Nevertheless, the concept of reduced platelet aggregation in response to activation in patients with a more severe bleeding phenotype is consistent with our results of reduced TRAP-induced percent platelets with activated GPIIb-IIIa in patients with high bleeding scores (Figure 3; supplemental Table 2). Similar to the nonsignificant trends we observe here (Figure 2), van Bladel et al 7 reported decreased ADP-induced platelet surface P-selectin measured by flow cytometry in patients with higher bleeding scores.…”

“…Like previous studies of pediatric ITP, 6,7,29 limitations of the present study include small sample size, few subjects with high bleeding scores, and variation in pharmacologic treatment. However, our finding of platelet tests associated with bleeding scores independent of platelet count in consecutively enrolled ITP patients regardless of treatment suggests that a larger study with more patients with higher bleeding scores and more uniform treatment would yield an even more robust result.…”

“…A likely explanation for the difference in findings is that we used whole blood flow cytometry to study platelet activation whereas Panzer et al 6,29 performed flow cytometric analysis on platelet-rich plasma, the preparation of which is prone to introduce artifactual platelet activation. 8 In a recent study, 7 van Bladel et al reported that pediatric chronic ITP patients with a severe bleeding phenotype demonstrated decreased platelet co-aggregation with normal donor platelets in response to phorbol myristate acetate, compared with patients with a mild bleeding phenotype. The aggregation response of normal donor platelets by the novel platelet aggregation method used by van Bladel et al ranged widely, raising concerns that the procedures associated with their method may introduce variation.…”

“…However, there are scant data addressing the question of whether tests of platelet function can predict bleeding in patients with ITP, 6,7 because most tests of platelet function are affected by thrombocytopenia. However, platelet function measured by whole blood flow cytometry is largely unaffected by the platelet count because individual platelets are analyzed one at a time.…”

Increased Platelet-Monocyte Interaction in Stable COPD in the Absence of Platelet Hyper-Reactivity

“…Platelet activation and reactivity were measured by a flow cytometry-based assay that has previously been described and that is able to detect both platelet hyper-reactivity and thrombopathy in different clinical settings [26][27][28]. In short, whole-blood samples were stained with antibodies for the platelet identification marker CD61 and for 2 platelet activation markers.…”