A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.

Central Hematology Laboratories Institute of Medical Biochemistry and Laboratory Diagnostics of the General University Hospital and of the 1st Faculty of Medicine of Charles University 128 20 Prague Czech Republic

Centre for Thrombosis and Hemostasis Institute of Hematology and Blood Transfusion 128 20 Prague Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technicka 5 166 28 Prague Czech Republic

Department of Biochemistry Institute of Hematology and Blood Transfusion 128 20 Prague Czech Republic

Department of Immunomonitoring and Flow Cytometry Institute of Hematology and Blood Transfusion 128 20 Prague Czech Republic

Laboratory for Disorders in Hemostasis Institute of Hematology and Blood Transfusion 128 20 Prague Czech Republic

See more in PubMed

Palma-Barqueros V., Revilla N., Sánchez A., Zamora Cánovas A., Rodriguez-Alén A., Marín-Quílez A., González-Porras J.R., Vicente V., Lozano M.L., Bastida J.M., et al. Inherited Platelet Disorders: An Updated Overview. Int. J. Mol. Sci. 2021;22:4521. doi: 10.3390/ijms22094521. PubMed DOI PMC

Al-Huniti A., Kahr W.H.A. Inherited Platelet Disorders: Diagnosis and Management. Transfus. Med. Rev. 2020;34:277–285. doi: 10.1016/j.tmrv.2020.09.006. PubMed DOI

Lambert M.P. Inherited Platelet Disorders: A Modern Approach to Evaluation and Treatment. Hematol. Clin. 2019;33:471–487. doi: 10.1016/j.hoc.2019.01.008. PubMed DOI

Noris P., Pecci A. Hereditary thrombocytopenias: A growing list of disorders. Hematol. Am. Soc. Hematol. Educ. Progr. 2017;2017:385–399. doi: 10.1182/asheducation-2017.1.385. PubMed DOI PMC

Greinacher A., Eekels J.J.M. Diagnosis of hereditary platelet disorders in the era of next-generation sequencing: “primum non nocere”. J. Thromb. Haemost. 2019;17:551–554. doi: 10.1111/jth.14377. PubMed DOI

Fasulo M.R., Biguzzi E., Abbattista M., Stufano F., Pagliari M.T., Mancini I., Gorski M.M., Cannavò A., Corgiolu M., Peyvandi F., et al. The ISTH Bleeding Assessment Tool and the risk of future bleeding. J. Thromb. Haemost. 2018;16:125–130. doi: 10.1111/jth.13883. PubMed DOI

Pecci A., Balduini C.L. Inherited thrombocytopenias: An updated guide for clinicians. Blood Rev. 2021;48:100784. doi: 10.1016/j.blre.2020.100784. PubMed DOI

Bolton-Maggs P.H.B., Chalmers E.A., Collins P.W., Harrison P., Kitchen S., Liesner R.J., Minford A., Mumford A.D., Parapia L.A., Perry D.J., et al. A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. Br. J. Haematol. 2006;135:603–633. doi: 10.1111/j.1365-2141.2006.06343.x. PubMed DOI

Lambert M.P. Updates in diagnosis of the inherited platelet disorders. Curr. Opin. Hematol. 2020;27:333–340. doi: 10.1097/MOH.0000000000000604. PubMed DOI

Harrison P., Mackie I., Mumford A., Briggs C., Liesner R., Winter M., Machin S. Guidelines for the laboratory investigation of heritable disorders of platelet function. Br. J. Haematol. 2011;155:30–44. doi: 10.1111/j.1365-2141.2011.08793.x. PubMed DOI

Rodeghiero F., Pabinger I., Ragni M., Abdul-Kadir R., Berntorp E., Blanchette V., Bodó I., Casini A., Gresele P., Lassila R., et al. Fundamentals for a Systematic Approach to Mild and Moderate Inherited Bleeding Disorders: An EHA Consensus Report. HemaSphere. 2019;3:e286. doi: 10.1097/HS9.0000000000000286. PubMed DOI PMC

Gresele P. Diagnosis of inherited platelet function disorders: Guidance from the SSC of the ISTH. J. Thromb. Haemost. 2015;13:314–322. doi: 10.1111/jth.12792. PubMed DOI

Megy K., Downes K., Morel-Kopp M.-C., Bastida J.M., Brooks S., Bury L., Leinoe E., Gomez K., Morgan N.V., Othman M., et al. GoldVariants, a resource for sharing rare genetic variants detected in bleeding, thrombotic, and platelet disorders: Communication from the ISTH SSC Subcommittee on Genomics in Thrombosis and Hemostasis. J. Thromb. Haemost. 2021;19:2612–2617. doi: 10.1111/jth.15459. PubMed DOI PMC

Elbaz C., Sholzberg M. An illustrated review of bleeding assessment tools and common coagulation tests. Res. Pract. Thromb. Haemost. 2020;4:761–773. doi: 10.1002/rth2.12339. PubMed DOI PMC

Rodeghiero F., Tosetto A., Abshire T., Arnold D.M., Coller B., James P., Neunert C., Lillicrap D. ISTH/SSC bleeding assessment tool: A standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J. Thromb. Haemost. 2010;8:2063–2065. doi: 10.1111/j.1538-7836.2010.03975.x. PubMed DOI

Elbatarny M., Mollah S., Grabell J., Bae S., Deforest M., Tuttle A., Hopman W., Clark D.S., Mauer A.C., Bowman M., et al. Normal range of bleeding scores for the ISTH-BAT: Adult and pediatric data from the merging project. Haemophilia. 2014;20:831–835. doi: 10.1111/hae.12503. PubMed DOI PMC

Gresele P., Orsini S., Noris P., Falcinelli E., Alessi M.C., Bury L., Borhany M., Santoro C., Glembotsky A.C., Cid A.R., et al. Validation of the ISTH/SSC bleeding assessment tool for inherited platelet disorders: A communication from the Platelet Physiology SSC. J. Thromb. Haemost. 2020;18:732–739. doi: 10.1111/jth.14683. PubMed DOI

Cattaneo M., Cerletti C., Harrison P., Hayward C.P.M., Kenny D., Nugent D., Nurden P., Rao A.K., Schmaier A.H., Watson S.P., et al. Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH. J. Thromb. Haemost. 2013 doi: 10.1111/jth.12231. PubMed DOI

Rubak P., Nissen P.H., Kristensen S.D., Hvas A.-M. Investigation of platelet function and platelet disorders using flow cytometry. Platelets. 2016;27:66–74. doi: 10.3109/09537104.2015.1032919. PubMed DOI

Gunning W.T., 3rd, Raghavan M., Calomeni E.P., Turner J.N., Roysam B., Roysam S., Smith M.R., Kouides P.A., Lachant N.A. A Morphometric Analysis of Platelet Dense Granules of Patients with Unexplained Bleeding: A New Entity of Delta-Microgranular Storage Pool Deficiency. J. Clin. Med. 2020;9:1734. doi: 10.3390/jcm9061734. PubMed DOI PMC

Zaninetti C., Greinacher A. Diagnosis of Inherited Platelet Disorders on a Blood Smear. J. Clin. Med. 2020;9:539. doi: 10.3390/jcm9020539. PubMed DOI PMC

Hayward C.P.M., Harrison P., Cattaneo M., Ortel T.L., Rao A.K. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function. J. Thromb. Haemost. 2006;4:312–319. doi: 10.1111/j.1538-7836.2006.01771.x. PubMed DOI

van Asten I., Schutgens R.E.G., Baaij M., Zandstra J., Roest M., Pasterkamp G., Huisman A., Korporaal S.J.A., Urbanus R.T. Validation of flow cytometric analysis of platelet function in patients with a suspected platelet function defect. J. Thromb. Haemost. 2018;16:689–698. doi: 10.1111/jth.13952. PubMed DOI

Ferrari S., Lombardi A.M., Putti M.C., Bertomoro A., Cortella I., Barzon I., Girolami A., Fabris F. Spectrum of 5’UTR mutations in ANKRD26 gene in patients with inherited thrombocytopenia: C.-140C>G mutation is more frequent than expected. Platelets. 2017;28:621–624. doi: 10.1080/09537104.2016.1267337. PubMed DOI

Noris P., Favier R., Alessi M.-C., Geddis A.E., Kunishima S., Heller P.G., Giordano P., Niederhoffer K.Y., Bussel J.B., Podda G.M., et al. ANKRD26-related thrombocytopenia and myeloid malignancies. Blood. 2013;122:1987–1989. doi: 10.1182/blood-2013-04-499319. PubMed DOI

Perez Botero J., Dugan S.N., Anderson M.W. ANKRD26-Related Thrombocytopenia. In: Adam M.P., Mirzaa G.M., Pagon R.A., Wallace S.E., Bean L.J.H., Gripp K.W., Amemiya A., editors. GeneReviews® [Internet] University of Washington, Seattle; Seattle, WA, USA: 1993–2022. [(accessed on 25 October 2022)]. Available online: https://www.ncbi.nlm.nih.gov/books/NBK507664/ PubMed

Galera P., Dulau-Florea A., Calvo K.R. Inherited thrombocytopenia and platelet disorders with germline predisposition to myeloid neoplasia. Int. J. Lab. Hematol. 2019;41((Suppl. S1)):131–141. doi: 10.1111/ijlh.12999. PubMed DOI

Arber D.A., Orazi A., Hasserjian R., Thiele J., Borowitz M.J., Le Beau M.M., Bloomfield C.D., Cazzola M., Vardiman J.W. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127:2391–2405. doi: 10.1182/blood-2016-03-643544. PubMed DOI

Khoriaty R., Ozel A.B., Ramdas S., Ross C., Desch K., Shavit J.A., Everett L., Siemieniak D., Li J.Z., Ginsburg D. Genome-wide linkage analysis and whole-exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia. Br. J. Haematol. 2019;186:574–579. doi: 10.1111/bjh.15961. PubMed DOI PMC

Kunishima S., Kashiwagi H., Otsu M., Takayama N., Eto K., Onodera M., Miyajima Y., Takamatsu Y., Suzumiya J., Matsubara K., et al. Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia. Blood. 2011;117:5479–5484. doi: 10.1182/blood-2010-12-323691. PubMed DOI

Kashiwagi H., Kunishima S., Kiyomizu K., Amano Y., Shimada H., Morishita M., Kanakura Y., Tomiyama Y. Demonstration of novel gain-of-function mutations of αIIbβ3: Association with macrothrombocytopenia and glanzmann thrombasthenia-like phenotype. Mol. Genet. Genom. Med. 2013;1:77–86. doi: 10.1002/mgg3.9. PubMed DOI PMC

Morais S., Oliveira J., Lau C., Pereira M., Gonçalves M., Monteiro C., Gonçalves A.R., Matos R., Sampaio M., Cruz E., et al. αIIbβ3 variants in ten families with autosomal dominant macrothrombocytopenia: Expanding the mutational and clinical spectrum. PLoS ONE. 2020;15:e0235136. doi: 10.1371/journal.pone.0235136. PubMed DOI PMC

Romano A.A., Allanson J.E., Dahlgren J., Gelb B.D., Hall B., Pierpont M.E., Roberts A.E., Robinson W., Takemoto C.M., Noonan J.A. Noonan syndrome: Clinical features, diagnosis, and management guidelines. Pediatrics. 2010;126:746–759. doi: 10.1542/peds.2009-3207. PubMed DOI

Pluthero F.G., Di Paola J., Carcao M.D., Kahr W.H.A. NBEAL2 mutations and bleeding in patients with gray platelet syndrome. Platelets. 2018;29:632–635. doi: 10.1080/09537104.2018.1478405. PubMed DOI

Aarts C.E.M., Downes K., Hoogendijk A.J., Sprenkeler E.G.G., Gazendam R.P., Favier R., Favier M., Tool A.T.J., van Hamme J.L., Kostadima M.A., et al. Neutrophil specific granule and NETosis defects in gray platelet syndrome. Blood Adv. 2021;5:549–564. doi: 10.1182/bloodadvances.2020002442. PubMed DOI PMC

Tariq H., Perez Botero J., Higgins R.A., Medina E.A. Gray Platelet Syndrome Presenting With Pancytopenia, Splenomegaly, and Bone Marrow Fibrosis. Am. J. Clin. Pathol. 2021;156:253–258. doi: 10.1093/ajcp/aqaa229. PubMed DOI

Bastida J.M., Lozano M.L., Benito R., Janusz K., Palma-Barqueros V., Del Rey M., Hernández-Sánchez J.M., Riesco S., Bermejo N., González-García H., et al. Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders. Haematologica. 2018;103:148–162. doi: 10.3324/haematol.2017.171132. PubMed DOI PMC

Andres O., König E.-M., Althaus K., Bakchoul T., Bugert P., Eber S., Knöfler R., Kunstmann E., Manukjan G., Meyer O., et al. Use of Targeted High-Throughput Sequencing for Genetic Classification of Patients with Bleeding Diathesis and Suspected Platelet Disorder. Open Compan. J. Thromb. Haemost. 2018;2:e445–e454. doi: 10.1055/s-0038-1676813. PubMed DOI PMC

Johns M.B.J., Paulus-Thomas J.E. Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol. Anal. Biochem. 1989;180:276–278. doi: 10.1016/0003-2697(89)90430-2. PubMed DOI

Kalina T., Flores-Montero J., van der Velden V.H.J., Martin-Ayuso M., Böttcher S., Ritgen M., Almeida J., Lhermitte L., Asnafi V., Mendonça A., et al. EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia. 2012;26:1986–2010. doi: 10.1038/leu.2012.122. PubMed DOI PMC

Chen D., Uhl C.B., Bryant S.C., Krumwiede M., Barness R.L., Olson M.C., Gossman S.C., Erdogan Damgard S., Gamb S.I., Cummins L.A., et al. Diagnostic laboratory standardization and validation of platelet transmission electron microscopy. Platelets. 2018;29:574–582. doi: 10.1080/09537104.2018.1476682. PubMed DOI