title: Measurement of platelet aggregation, independently of patient platelet count: a flow-cytometric approach.
abstract: Essentials Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. A flow cytometric platelet aggregation test independent of the patient platelet count was made. Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer. High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients.
  
  
  
  Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count, and examine the association of aggregation with a bleeding history in thrombocytopenic patients. Patients/methods We established a flow-cytometric assay of platelet aggregation, and measured samples from healthy individuals preincubated with antiplatelet drugs, and samples from two patients with inherited platelet disorders. Then, we included 19 healthy individuals and 20 patients with platelet counts of ≤ 50 × 109L-1, diagnosed with acute myeloid leukemia or myelodysplastic syndrome. We measured platelet aggregation and platelet activation by platelet surface expression of activated glycoprotein IIb-IIIa, P-selectin and CD63 after addition of agonists: collagen-related peptide, thrombin receptor-activating peptide (TRAP), and ADP. Results The platelet aggregation assay showed a low intraserial coefficient of variation of ≤ 3%. Similar results were obtained for platelet-rich plasma and isolated platelets at platelet counts of > 10 × 109L-1; otherwise, platelet isolation was required. The platelet aggregation percentage decreased with increasing antiplatelet drug concentration. Platelet aggregation in patients was reduced as compared with healthy individuals: 42% (interquartile range [IQR] 27-58) versus 66% (IQR 60-67) for TRAP; 41% (IQR 25-48) versus 70% (IQR 69-72) for collagen-related peptide; and 44% (IQR 30-53) versus 65% (IQR 46-72) for ADP. Platelet activation after stimulation was reduced in patients and correlated with platelet aggregation (e.g. r = 0.78-0.81 when stimulated with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark.
authors.forename: P J
authors.identifier: 0000-0002-2035-0169
authors.initials: PJ
authors.lastname: Vinholt
authors.affiliation: Department of Hematology, Odense University Hospital, Odense, Denmark.
authors.forename: H
authors.initials: H
authors.lastname: Frederiksen
authors.affiliation: Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: A-M
authors.initials: AM
authors.lastname: Hvas
authors.affiliation: Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
authors.forename: U
authors.initials: U
authors.lastname: Sprogøe
authors.affiliation: Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
authors.forename: C
authors.initials: C
authors.lastname: Nielsen
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis; D000293:Adolescent; D000328:Adult; D000368:Aged; D002352:Carrier Proteins / Q000097:blood; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006470:Hemorrhage / Q000175:diagnosis; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000097:blood; D008297:Male; D008875:Middle Aged; D009190:Myelodysplastic Syndromes / Q000097:blood; D019007:P-Selectin / Q000378:metabolism; D010455:Peptides / Q000097:blood; D010974:Platelet Aggregation / Q000187:drug effects*; D010976:Platelet Count; D010979:Platelet Function Tests / Q000379:methods*; D012016:Reference Values; D015203:Reproducibility of Results; D012306:Risk; D013921:Thrombocytopenia / Q000097:blood / Q000175:diagnosis; D055815:Young Adult
chemical_list: D002352:Carrier Proteins; D019007:P-Selectin; D010455:Peptides; C546937:SELP protein, human; C435908:collagen-related peptide; D000244:Adenosine Diphosphate
keywords: flow cytometry; hematologic neoplasms; methods; platelet aggregation; thrombocytopenia