Electron microscopy examination of platelet whole mount preparations to quantitate platelet dense granule numbers: Implications for diagnosing suspected platelet function disorders due to dense granule deficiency.

INTRODUCTION: Dense granule (DG) deficiency (DGD) is a feature of some platelet function disorders (PFD) with a prevalence similar to von Willebrand disease. Most laboratories assess for DGD using whole mount platelet preparations and electron microscopy (EM). We evaluated our experiences with this test and associations between DGD and bleeding. METHODS: Dense granule EM records for 2006-2017 were examined for patients and simultaneously tested controls, and for an overlapping PFD study cohort to evaluate findings and their relationship to bleeding. RESULTS: More patient than control samples had reduced DG counts (6.5% vs 0.3%, P < .01). DG counts showed no relationship to age or mean platelet volume and had acceptable within-subject variability that was higher for DGD than control participants (28% vs 12%). Repeat tests confirmed DGD in all persons with initial DG counts <4.0/platelet, but not in those with less severe reductions (4.0-4.8 DG/platelet) or normal DG counts (≥4.9 DG/platelet). Aggregometry and adenosine triphosphate release tests, respectively, had only ~52% and 70% sensitivity for DGD. Confirmed DGD by EM was associated with higher bleeding scores and a bleeding disorder. CONCLUSION: Whole mount EM is useful for the evaluation of suspected PFD due to DGD and detects abnormalities associated with bleeding.

Molecular phenotype and bleeding risks of an inherited platelet disorder in a family with a RUNX1 frameshift mutation.

INTRODUCTION: Inherited defects in RUNX1 are important causes of platelet function disorders. AIM: Our goals were to evaluate RUNX1-related platelet disorders among individuals evaluated for uncharacterized, inherited platelet function disorders and test a proof of concept that bleeding risks could be quantitatively estimated for typical families with an inherited platelet function disorder. METHODS: Index cases with an uncharacterized inherited platelet function disorder were subjected to exome sequencing with confirmation of RUNX1 mutations by Sanger sequencing. Laboratory findings were obtained from medical records and persistence of platelet non-muscle myosin heavy chain IIB (MYH10), a biomarker of RUNX1 defects, was assessed by Western blotting. Bleeding histories were assessed using standardized assessment tools. Bleeding risks were estimated as odds ratios (OR) using questionnaire data for affected individuals compared to controls. RESULTS: Among 12 index cases who had their exomes sequenced, one individual from a family with eight study participants had a c.583dup in RUNX1 that segregated with the disease and was predicted to cause a frameshift and RUNX1 haploinsufficiency. Unlike unaffected family members (n = 2), affected family members (n = 6) had increased bleeding scores and abnormal platelet aggregation and dense granule release responses to agonists but only some had thrombocytopenia and/or dense granule deficiency. This family's mutation was associated with persistence of MYH10 in platelets and increased risks (OR 11-440) for wound healing problems and mild bleeding symptoms, including bleeding interfering with lifestyle in women. CONCLUSION: Inherited platelet dysfunction due to a RUNX1 haploinsufficiency mutation significantly increases bleeding risks.

Variability in platelet dense granule adenosine triphosphate release findings amongst patients tested multiple times as part of an assessment for a bleeding disorder.

INTRODUCTION: Lumi-aggregometry quantification of platelet dense granule adenosine triphosphate (ATP) release is commonly used for diagnosing platelet function disorders. As the test findings show considerable variability for healthy controls, we postulated that patient findings might also be variable and investigated patients who were assessed for dense granule ATP release defects more than once. METHODS: Analyses were performed on prospectively collected data for first and second tests for subjects tested for dense granule ATP release defects more than once by the Hamilton Regional Laboratory Program (HRLMP) between January 2007 and June 2013 (cohort I). Similar analyses were performed for subjects who were recruited to a platelet disorder study (cohort II) and were assessed for ATP release defects more than once before October 2015. RESULTS: A total of 150 unique subjects had multiple ATP release tests. Results with individual agonists were variable for many subjects. While normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), impaired release with multiple agonists was confirmed in only some subjects (cohort I: 34%; cohort II: 54%). Inconsistent findings were common (cohort I: 36%; cohort II: 39%). ISTH bleeding scores showed no relationship to the test findings. The finding of impaired ATP release with 2 or more agonists on both tests was not associated with an increased likelihood of a definite bleeding disorder. CONCLUSION: The variability in platelet dense granule ATP release findings amongst patients assessed for diagnostic purposes suggests that the test has limited value for diagnosing platelet disorders.