Platelet dense granule production and dense granule release defects in pediatric patients with storage pool disorder

J. Eilenberger1, G. Manukjan1, O. Andres1, C. Schambeck2, S. Eber2, H. Schulze1 (1W├╝rzburg, Germany, 2Munich, Germany)

Platelets - Physiology and Disorders of platelet number and function
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: Storage pool disease (SPD) comprises a group of platelet defects where alpha and/or delta granules are reduced or cannot be secreted in response to agonists. The detection of delta granule release has been hampered as there are no simple assays. We aim to implement a kinetic mepacrine assay to better identify the pathomechanism in children with a dense granule-based bleeding diathesis.

Methods: We developed a flow cytometric mepacrine assay which measures the unstained platelets, determines the mepacrine uptake and finally the decline in fluorescence after agonist stimulation over 5 minutes. The analysis was performed using FlowJo software. We correlated the mean fluorescence intensity of mepacrine uptake and release patterns with CD63 exposure, ATP release and the ADP/ATP ratio. The bleeding score was assessed by the translated ISTH-BAT. We analyzed 56 children with SPD whose initial diagnosis was confirmed in a second site visit.

Results: 90% of children had a positive family history of bleedings, mostly with the mother also suffering from prolonged bleeding epiosdes. Most children had an ISTH-BAT score of 2, 3 or 4. The delta granule release (ADP and ATP) was significantly reduced in our cohort with 75% having values below the reference threshold value of 10.1 umol/10e12 platelets. The mepacrine release was statistically reduced in patients with SPD compared to controls (p>0.05). The ability to release mepacrine in response to TRAP correlated with the delta-granule release measure by ADP+ATP release. We detected a reduced CD63 expression by flow cytometry in response to ADP or TRAP-6 in 12 children. 6 patients showed defective mepacrine uptake, 7 a selective delta granule release, 13 had a combined uptake and release defect. 10 patients showed a normal uptake and release, when compared to healthy adult controls. The overall response to ADP and TRAP in aggregometry and flow cytometry correlated well, indicating that the underlying defect is a granule-based defect and not due to an agonist receptor.

Conclusion: Our data show that implementation of a time-resolved mepacrine iallows to distinguish mepacrine uptake, mepacrine release, and combined defects. It is a quick and inexpensive tool that can readily be added to help refining the diagnosis.