Characterization of Gray Platelet Syndrome by Flow Cytometry
D Chen, RS Miller, MM Patnaik, RK Pruthi, MA Elliott, JA Heit, WG Owen, WL Nichols. Mayo Clinic, Rochester, MN
Background: Gray platelet syndrome (GPS) is a rare hereditary platelet disorder defined by platelet α-granule deficiency. Patients with GPS usually have mild-moderate bleeding tendency. Laboratory diagnosis of GPS is primarily based on peripheral blood (PB) smear light microscopy and electron microscopic examination. The goal of this study is to establish a flow cytometry method to indirectly detect platelet α-granule deficiency by measuring surface expression of an α-granule membrane protein, P-selectin, following platelet activation.
Design: A total of 4 patients diagnosed with GPS were identified from Mayo Clinic platelet disorder database. Their clinical/family histories were reviewed. CBC and PB smears were examined. Coagulation tests for bleeding disorders were performed on patients' citrated plasma samples, including PT, APTT, TT, von Willebrand factor, factor VIII, IX and XIII activities. Platelet aggregation and electron microscopy studies were performed on platelet rich plasma. Flow cytometry studies were performed using citrated whole blood samples from all 4 patients and 55 normal donors. Platelet surface glycoproteins were measured using fluorochrome-conjugated antibodies against GPIIb, GPIIIa, GPIX and GPIbα. Platelet surface expression of P-selectin and activated GPIIb-IIIa, following stimulation by 10μM thrombin receptor activating peptide (TRAP) and 10μM ADP were measured using fluorochrome-conjugated anti-P-selectin and PAC-1 antibody, respectively.
Results: All four patients (3 female, 1 male, median age 26 at diagnosis) had thrombocytopenia (12∼63 x10(9)/L) and well documented personal and family bleeding history. They all had unremarkable plasmatic hemostatic testing results. Platelets on PB smears appeared markedly hypogranular. Two patients had normal platelet aggregation responses and 2 patient showed abnormal responses to all stimuli. EM studies on patients' platelets showed markedly decreased α-granules. Platelets from all 4 patients showed normal surface expression of glycoproteins IIb, IIIa, Ibα and IX. However, in comparison with normal donor platelets, they all had markedly decreased P-selectin expression despite normal PAC-1 binding upon stimulation.
Conclusions: The markedly decreased platelet α-granules in GPS can be indirectly detected by measuring P-selectin expression upon platelet activation. This flow cytometric method, although still needing further investigation and standardization, could potentially be a surrogate laboratory test for diagnosing or confirming GPS.
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 206, Wednesday Afternoon