Flow cytometry immunophenotyping of healthy platelets and hospitalized patients with suspected platelet dysfunction: challenges for establishing a cutoff value

Introduction/Objective: Platelets play an essential role in hemostasis, for their characterization, several platelet surface glycoproteins (GP) were identified as GPIIb, evaluated by the expression of CD41, and non-covalently binds to GPIIIa, recognized by the CD61 marker, making up the GPIIb-GPIIIa complex (CD41/CD61). GPIX is identified by CD42a and GPIb by CD42b that interacts together with GPV (CD42d), forming the other complex, GPIb-IX-V. Flow Cytometry (FC) is one of the techniques which allows the identification and characterization of platelets. The detection of absent or reduced expression of the glycoproteins is the main objective of this technique. Abnormalities of glycoproteins lead to hemorrhagic syndromes. Among the main diseases, Bernard Soulier (BS) and Glanzmann's Thrombasthenia (GT) stand out. BS is an disease caused by several molecular changes that produce altered proteins that would have a fundamental role in the stability of the expression of the von Willebrand receptor GPIb/IX/V complex (CD42a/CD42b/CD42d). GT is an thrombocytopathy caused by deficiency or decrease in the fibrinogen receptor GPIIb-GPIIIa (CD41/CD61). We aimed to show a FC based platelet assessment test for diagnostic use, which measures the expression of markers in normal patients, and evaluate these markers in patients with platelet disorders. Materials and methods: We examined a control group of 41 healthy adults to establish reference values and assess the variability of the relative expression of platelet markers and compare to 30 patients with suspected platelet dysfunctions. Were determined the MFI of the expressed parameters by FC using CD41, CD42a, CD42b and CD61, and SSC/FSC platelet gated cells. Results: We determined our baseline panel of markers and compared them with suspected platelet dysfunctions. Patients with suspected BS presented increased levels of MFI for GPIIIa (CD61) and GPIIb (CD41). They showed significantly reduced levels of GPIb (CD42b) and GPIX (CD42a). Patients with suspected GT showed normal expression of GPIX (CD42a), increased expression of GPIb (CD42b) and reduced levels of GPIIIa (CD61), showing expression patterns like those described in the literature, which characterizes GT as deficiency or decrease of one of the markers of the GPIIb/GPIIIa complex (CD41/CD61). Discussion: We describe the FC assay to support the diagnosis of different platelet disorders. We standardized and implemented the technique in which the expression profile of platelet-associated immunophenotypic markers was determined in a population of hematological normal individuals. It was possible to incorporate the test into laboratory research, to assist in the study of various pathologies associated with platelet dysfunction through the evaluation of the expression of these markers. With the analyzes in our laboratory, it was possible to evaluate 30 patients with suspected platelet dysfunction and contribute to diagnostic elucidation. Conclusions: Our study made it possible to implement a technique that brought benefits to care. These analyzes can be implemented in clinical practice to improve diagnosis for patients with suspected platelet disorders.