Background: The method of immunofluorescence staining of blood smears is a recently developed (Greinacher et al. J Thromb Haemost 2017; 15: 1511-1521) approach of remote diagnostics of various platelet pathologies including MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombastenia, and gray platelet syndrome, and others. We report here experience of introducing this method in the National Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev (Moscow, Russia), which is the main national pediatric hematology hospital that provides diagnosis and therapy to children with blood disorders throughout the country.

Aims: Our study aimed to transfer this relatively labor-intensive and skill-sensitive methodology, to introduce in the routine laboratory practice, and to perform its validation.

Methods: Blood smears from healthy donors and patients were prepared using the standard method established worldwide, air-dried, and storage. Citrate anticoagulated blood was used for preparation. For immunofluorescence labeling was used primary antibodies (Myosin, LAMP 1 (H5G11), LAMP 2 (H4B4), VWF, P-Selectin (CD62P), CD63 (delta-granules), Ib/IX CD42a (FMC25), IIb/IIIa CD41 P2, β1-Tubulin (2.1.), α-Tubulin (RM113)) and fluorescence labeling secondary antibodies. Blood smears were assessed by immunofluorescence microscopy.

Results: Patients with different platelet disorders (9 patients with MYH9 disorder, 7 patients with Wiskott-Aldrich syndrome, 2 patients with Bernard-Soulier syndrome, 2 patients Hermansky-Pudlak syndrome,1 patient with gray platelets syndrome, 1 patient with Glanzmann thrombastenia) confirmed by the genetic analysis and clinical symptoms were assessed. In addition to the immunofluorescence assay, each patient was analyzed platelet function by flow cytometry and aggregation by standard methods. For all patients, we were able to identify the abnormalities by immunofluorescence associated with established diagnosis.
The study was supported by the grant of the President of the Russian Federation.

Conclusions: In this work it was determined that the immunofluorescence staining of blood smears enables us to receive reproducible results and implement the diagnostic of platelet disorders.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/immunofluorescence-staining-of-blood-smears-for-the-diagnostics-of-platelet-disorders-a-single-center-experience-in-a-pediatric-hospital/