Platelet Transmission Electron Microscopy and Flow Cytometry in the Diagnosis of Congenital/Hereditary Qualitative or Quantitative Platelet Disorders

Abstract

BackgroundCongenital/hereditary qualitative platelet disorders (CQPD) can be classified by platelet count (eg, normal or decreased) and platelet size as well as by functional defect. Based on mean platelet volume (MPV), congenital thrombocytopenias (CT) are classified as macro-thrombocytopenias (eg, Bernard-Soulier syndrome [BSS], MYH-9 [myosin heavy chain 9, non-muscle]-associated thrombocytopenias, gray platelet syndrome [GPS], etc.); microthrombocytopenias (eg, Wiskott-Aldrich syndrome and variants); and those with normal platelet size. CQPDs with normal platelet count include Glanzmann thrombasthenia (GT), dense granule storage pool disorders (DG-SPD) including Hermansky-Pudlak syndrome (HPS), platelet secretion disorders and many others. Diagnosis has relied on platelet counting and sizing, blood smear light microscopy, platelet aggregation (agg) and platelet function analyzer (PFA-100) studies. Platelet transmission electron microscopy (PTEM) and flow cytometry can provide supplementary information and molecular analysis is also evolving. The objective of this study was to determine the roles of platelet transmission electron microscopy (PTEM) and flow cytometry in the diagnosis of CQPD and CT.MethodsIn this retrospective cohort study, after IRB approval, the electronic medical record system was queried for patients (pt) with a confirmed CQPD or CT seen at Mayo Clinic between 2000 and 2015. A detailed chart review was undertaken.Results54 pt (64% female) met our study criteria; median age was 32 years (range 1 day to 81 years). 18/54 (33%) pt had macrothrombocytopenia: BSS 4 (7%); MYH-9 10 (19%); gray platelet syndrome 4 (7%). 36/54 (66%) had normal MPV: Glanzmann thrombasthenia 6 (11%); storage pool disorders including DG-SPD 10 (19%) and HPS 2 (3%), mild alpha granule deficiency 3 (6%) and York platelet syndrome 4 (7%); platelet secretion disorders 4 (7%); ANKRD26 mutation 3 (6%); congenital amegakaryocytic thrombocytopenia 1 (2%); GATA-1 mutation 1 (2%); RUNX-1 mutation 1 (2%); and Jacobsen syndrome 1 (2%). 44 pt (81%) had a positive bleeding history: epistaxis 52%, cutaneous bleeding 57%, gastrointestinal bleeding 21% and menorrhagia 54%; 10 pt (19%) had a negative bleeding history. Results of standard and esoteric platelet testing are summarized in Table 1.Diagnosis in the 10 pt with glycoprotein deficiency (BSS or GT) was established by agg; flow cytometry was confirmatory but not necessary. Of the 23 pt with SPD, 5 had a negative bleeding history; 4 pt had normal agg and PFA-100 and were diagnosed exclusively by PTEM. In this group, the abnormalities on platelet aggregation and PFA were variable and PTEM allowed for a better definition of the specific abnormalities. For MYH-9 patients, PTEM confirmed leukocyte inclusions that had already been identified on light microscopy. PTEM was the only diagnostic modality able to identify the abnormality in York platelet syndrome. In 5/54 (9%) genetic testing was necessary for diagnosis (GATA-1, ANKRD26 and RUNX-1)ConclusionIn this cohort, standard platelet assays established the diagnosis in the large majority of CQPD and CT. PTEM was essential for confirmation in DG-SPD, mild alpha granule deficiency and York platelet syndrome and useful in combination with molecular testing to establish a diagnosis in selected cases. Guidance on selection of patients for such specialized testing requires further study.Table 1Results of standard and esoteric platelet testing for 54 patients with congenital qualitative platelet disorders and/or thrombocytopeniaDisordern (%)Platelet aggregationPFAPTEMFlow cytometryConfirmation by geneticsDoneAbnormalDoneAbnormalDoneAbnormalDoneAbnormalGlanzmann thrombasthenia6 (11)665510330Bernard-Soulier syndrome & variants4 (7)444430440MYH-9 related disorders10 (19)728355425Dense granule deficiency and HPS12 (22)1281281212400Alpha granule deficiency*7 (13)747677441York platelet syndrome4 (7)101144110TXA2 or PG synthesis/receptor defect4 (7)444330300CATM1 (2)0-0-0-0-1ANKRD263 (6)333233333GATA-11 (2)0-1111111RUNX-11 (2)10110-0-1Jacobsen syndrome1 (2)101111101MYH-9: myosin heavy chain 9, non-muscle (MHY-9) associated thrombocytopenia, HPS: Hermansky-Pudlak Syndrome, TXA2: thromboxane A2, PG: prostaglandin CATM: congenital amegakaryocytic thrombocytopenia. * Includes gray platelet syndrome DisclosuresPardanani:Stemline: Research Funding.