[Developments of high-throughput sequencing-based diagnosis of congenital thrombocytopenia/platelet disorders in a registry study].

Congenital thrombocytopenia/platelet disorders are heterogeneous disorders of platelet number and/or function. Pathogenic variants in the genes implicated in megakaryocyte differentiation and platelet formation cause thrombocytopenia in these patients. Recent advances have elucidated several causative genes for these disorders, but identifying the underlying causative genes remains challenging. Patients with these disorders often receive inappropriate treatments, including glucocorticoids and splenectomy, for chronic immune thrombocytopenia (ITP). In Japan, we have developed a diagnostic system using high-throughput DNA sequencing with a multigene panel and established a registry. Between 2018 and 2023, 245 patients were enrolled and analyzed. Pathogenic variants in 17 genes (42 MYH9, 19 ANKRD26, 17 ITGA2B/ITGB3, 8 ACTN1, 8 WAS, 6 ETV6, 6 VWF, 5 CYCS, and 14 others) were identified in 125 patients (51.0%). An additional 29 patients (11.8%) had suspected pathogenic variants under investigation. We also found that immature platelet fraction (IPF%) is useful in the differential diagnosis because the median IPF% in MYH9 disorders, 48.7%, was significantly higher than in all other groups (chronic ITP, 13.4%; controls, 2.6%). The results of this study provide new insight into congenital thrombocytopenia/platelet disorders.

Comprehensive genetic analysis for identification of monogenic disorders and selection of appropriate treatments in pediatric patients with persistent thrombocytopenia.

Persistent thrombocytopenia is caused by various diseases, including immune thrombocytopenia, inherited thrombocytopenia, and inherited bone marrow failure syndromes. Considering the large number of genes responsible for inherited disorders, comprehensive genetic analysis is required to diagnose monogenic disorders. In this study, we enrolled 53 pediatric patients with persistent thrombocytopenia exhibiting visually small or normal-sized platelets. We performed whole-exome sequencing, including 56 genes responsible for inherited thrombocytopenia, and evaluated clinical parameters according to disease type. Among 53 patients, 12 patients (22.6%) were diagnosed with monogenic disorders. Nine patients had a family history of thrombocytopenia. Pathogenic or novel variants of genes responsible for inherited thrombocytopenia were identified in three and six patients, respectively. The variants in genes for inherited thrombocytopenia with large or giant platelets were unexpectedly identified in six patients. Pathogenic variants in genes for inherited bone marrow failure syndromes with systemic features were identified in three patients with atypical symptoms. Since the definitive diagnostic methods for immune thrombocytopenia are limited, and a substantial number of patients with inherited thrombocytopenia are at a high risk of developing malignancies, comprehensive genetic analysis is indispensable for selecting appropriate therapies, avoidance of unnecessary treatments for immune thrombocytopenia, and long-term follow-up of patients with inherited thrombocytopenia.

MYH9-related inherited thrombocytopenia: the genetic spectrum, underlying mechanisms, clinical phenotypes, diagnosis, and management approaches.

Inherited thrombocytopenias have been considered exceedingly rare for a long time, but recent advances have facilitated diagnosis and greatly enabled the discovery of new causative genes. MYH9-related disease (MYH9-RD) represents one of the most frequent forms of inherited thrombocytopenia, usually presenting with nonspecific clinical manifestations, which renders it difficult to establish an accurate diagnosis. MYH9-RD is an autosomal dominant-inherited thrombocytopenia caused by deleterious variants in the MYH9 gene encoding the heavy chain of nonmuscle myosin IIA. Patients with MYH9-RD usually present with thrombocytopenia and platelet macrocytosis at birth or in infancy, and most of them may develop one or more extrahematologic manifestations of progressive nephritis, sensorial hearing loss, presenile cataracts, and elevated liver enzymatic levels during childhood and adult life. Here, we have reviewed recent advances in the study of MYH9-RD, which aims to provide an updated and comprehensive summary of the current knowledge and improve our understanding of the genetic spectrum, underlying mechanisms, clinical phenotypes, diagnosis, and management approaches of this rare disease. Importantly, our goal is to enable physicians to better understand this rare disease and highlight the critical role of genetic etiologic analysis in ensuring accurate diagnosis, clinical management, and genetic counseling while avoiding ineffective and potentially harmful therapies for MYH9-RD patients.

Impact of thrombocytopenia-associated c.-118C>T and c.-140C>G ANKRD26 5'UTR variants in three-generational pedigree.

Inherited thrombocytopenias (ITs) encompass a group of rare disorders characterized by diminished platelet count. Recent advancements have unveiled various forms of IT, with inherited thrombocytopenia 2 (THC2) emerging as a prevalent subtype associated with germline variants in the critical 5' untranslated region of the ANKRD26 gene. This region is crucial in regulating the gene expression of ANKRD26, particularly in megakaryocytes. THC2 is an autosomal dominant disorder presenting as mild-to-moderate thrombocytopenia with minimal symptoms, with an increased risk of myeloproliferative malignancies. In our study of a family with suspected IT, three affected individuals harbored the c.-118C>T ANKRD26 variant, while four healthy members carried the c.-140C>G ANKRD26 variant. We performed a functional analysis by studying platelet-specific ANKRD26 gene expression levels using quantitative real-time polymerase-chain reaction. Functional analysis of the c.-118C>T variant showed a significant increase in ANKRD26 expression in affected individuals, supporting its pathogenicity. On the contrary, carriers of the c.-140C>G variant exhibited normal platelet counts and no significant elevation in the ANKRD26 expression, indicating the likely benign nature of this variant. Our findings provide evidence confirming the pathogenicity of the c.-118C>T ANKRD26 variant in THC2 and suggest the likely benign nature of the c.-140C>G variant.

What is the context?Inherited thrombocytopenias (ITs) are rare conditions characterized by low platelet counts. Inherited thrombocytopenia 2 (THC2) is caused by ANKRD26 gene changes leading to increased ANKRD26 expression as the main reason for subsequent thrombocytopenia. THC2 results in a mild-to-moderate decrease in platelet count and increases blood cancer risk. We focused on understanding two ANKRD26 variants in a family with a history of thrombocytopenia.What is new?We conducted functional analysis to understand the effect of variants on platelet function and gene expression. We identified three thrombocytopenic family members as carriers of ANKRD26 variant c.-118C>T. This variant is linked to increased expression of the ANKRD26 gene and confirmed as the likely cause of THC2. Another variant, c.-140C>G, was present in four healthy family members. Although it was considered causal for THC2 in the past, our study suggests that the c.-140C>G variant does not elevate ANKRD26 expression and does not cause thrombocytopenia.What is the impact?Understanding the genetic and functional implications of ANKRD26 gene variants is crucial for THC2 diagnosis and management. Our study emphasizes the necessity of conducting functional analyses to precisely evaluate the clinical significance of variants linked to inherited blood disorders. Carriers of the c.-118C>T variant should undergo vigilant monitoring for THC2 and potential cancer development. Conversely, the c.-140C>G variant does not pose a risk of THC2 or heightened cancer susceptibility.

Thrombocytopenia 4 (THC4): Six novel families with mutations of the cytochrome c gene.

Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants.

Bernard-Soulier syndrome caused by two novel heterozygous GP1BA gene mutations: a case report and literature review.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a rare inherited macrothrombocytopenia, usually autosomal recessive, which is characterized by prolonged bleeding, thrombocytopenia, and abnormally large platelets. METHODS: For more than 6 years, we misdiagnosed a patient with BSS without an obvious bleeding tendency as having idiopathic thrombocytopenia purpura (ITP), prior to obtaining a genetic analysis. On admission, routine hematology showed a platelet count of 30 × 109/L and mean platelet volume (MPV) of 14.0 fL. RESULTS: Whole-exome sequencing revealed two likely pathogenic heterozygous mutations (c.95_101del and c.1012del) in GP1BA. Flow cytometry analysis of platelet membrane glycoproteins indicated that the expression of GP1b was 0.28% of the normal level. Platelet aggregation tests indicated that platelet aggregation was inhibited by ristocetin- (1.7%), ADP- (14.5%), and arachidonic acid- (5.6%) induced platelet aggregation. A literature review identified reports on 53 mutations in the GP1BA gene in 253 patients, 29 mutations in the GP1BB gene in 90 patients, and 32 mutations in the GP9 gene in 114 patients. CONCLUSION: This case report describes two novel gene mutation sites that have not been reported previously, enriching understanding of the GP1BA mutation spectrum.

Aggregates of nonmuscular myosin IIA in erythrocytes associate with GATA1- and GFI1B-related thrombocytopenia.

BACKGROUND: The transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. OBJECTIVES: To expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. METHODS: We assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. RESULTS: We investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 µm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. CONCLUSION: Aggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

Humanized mouse models for inherited thrombocytopenia studies.

Inherited thrombocytopenia (IT) is a group of hereditary disorders characterized by a reduced platelet count as the main clinical manifestation, and often with abnormal platelet function, which can subsequently lead to impaired hemostasis. In the past decades, humanized mouse models (HMMs), that are mice engrafted with human cells or genes, have been widely used in different research areas including immunology, oncology, and virology. With advances of the development of immunodeficient mice, the engraftment, and reconstitution of functional human platelets in HMM permit studies of occurrence and development of platelet disorders including IT and treatment strategies. This article mainly reviews the development of humanized mice models, the construction methods, research status, and problems of using humanized mice for the in vivo study of human thrombopoiesis.

Humanized mouse models (HMMs) refer to immunodeficient mice that have been used for the investigation of human hematopoiesis and immunity for years. With engrafted human hematopoietic stem cells (HSCs), the differentiation process of HSCs and re-construction of platelets can be monitored in the mice. Until now, several strains of HMMs have been used in the studies of inherited thrombocytopenia (IT), a genetic disorder associated with low platelet count in the blood. In this study, we reviewed the development of these HMMs in IT studies, compared the different sources of HSCs transplanted into HMMs and summarize the strategies of HSC transplantation in HMMs. The Kit^−/− immunodeficient mice showed effectively long-term and stable implantation of human HSC without irradiation and higher implantation levels, and they also support multilinear differentiation of human HSC, such as platelets and red blood cells. The source and count of HSCs and the transplantation strategy may also impact the result. This study provides a basis information for HMMs used in IT and will help other investigators in this field choosing the right research plan.

A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure.

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.

Novel SLFN14 mutation associated with macrothrombocytopenia in a patient with severe haemorrhagic syndrome.

BACKGROUND: Platelet-type bleeding disorder 20 (BDPLT20), as known as SLFN14-related thrombocytopenia, is a rare inherited thrombocytopenia (IT). Previously, only 5 heterozygous missense mutations in the SLFN14 gene have been reported. METHODS: A comprehensive clinical and laboratory examination of a 17-year-old female patient with macrothrombocytopenia and severe mucocutaneous bleeding was performed. Examination was carried out using standardized questionnaires to assess bleeding, high-throughput sequencing (Next Generation Sequencing), optical and fluorescence microscopy, flow cytometry with activation and analysis of intracellular calcium signaling of platelets, light transmission aggregometry and thrombus growth in the flow chamber. RESULTS: Analysis of the patient's genotype revealed a previously undescribed c.655 A > G (p.K219E) variant in the hotspot of the SLFN14 gene. Immunofluorescence and brightfield examination of platelets in the smear showed heterogeneity in cells size, including giant forms over 10 µm (normal size 1-5) in diameter, with vacuolization and diffuse distribution of ß1-tubulin and CD63. Activated platelets showed impaired contraction and shedding/internalization of GPIb. GP IIb/IIIa clustering was increased at rest and attenuated upon activation. Intracellular signalling study revealed impaired calcium mobilization upon TRAP 35.97 nM (reference range 180 ± 44) and CRP-XL 10.08 nM (56 ± 30) stimulation. Aggregation with ADP, collagen, TRAP, arachidonic acid and epinephrine was impaired in light transmission aggregometry; agglutination with ristocetin persisted. In the flow chamber with a shear rate of 400 s-1 platelet adhesion to collagen and clot growth were impaired. CONCLUSION: The revealed disorders of phenotype, cytoskeleton and intracellular signaling explain the nature of SLFN14 platelet dysfunction and the patient's severe hemorrhagic syndrome.

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation.

Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in GNE, SLC35A1, GALE and B4GALT is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.

Inherited bone marrow failure with macrothrombocytopenia due to germline tubulin beta class I (TUBB) variant.

Germline mutations in tubulin beta class I (TUBB), which encodes one of the ß-tubulin isoforms, were previously associated with neurological and cutaneous abnormalities. Here, we describe the first case of inherited bone marrow (BM) failure, including marked thrombocytopenia, morphological abnormalities, and cortical dysplasia, associated with a de novo p.D249V variant in TUBB. Mutant TUBB had abnormal cellular localisation in transfected cells. Following interferon/ribavirin therapy administered for transfusion-acquired hepatitis C, severe pancytopenia and BM aplasia ensued, which was unresponsive to immunosuppression. Acquired chromosome arm 6p loss of heterozygosity was identified, leading to somatic loss of the mutant TUBB allele.

A family case series of inherited thrombocytopenia.

Inherited thrombocytopenia (IT) is a heterogeneous group of diseases with a genetic origin. The primary symptom presented by patients is a reduced platelet count in the peripheral blood. Nevertheless, certain forms of IT are characterized by the occurrence of other congenital malformations or predisposition to acquire additional diseases. Five related subjects with lifelong thrombocytopenia were admitted to our clinic. A total of 16 cases of persistent thrombocytopenia were investigated in the family history. Molecular and cytogenetic analysis covered MECOM, MPL, RUNX1, ETV6, and GATA1 genes, whose mutations are known to cause predisposing forms of IT. The laboratory testing revealed thrombocytopenia ranging from 19 to 65 × 109/L in the subjects. Mild bleeding symptoms were present in each of the subjects, while two of five had a history of severe hemorrhage requiring transfusion of blood products. Establishing a diagnosis of IT protects the patient from unnecessary treatment and enables the appropriate surveillance.

"CHildren with Inherited Platelet disorders Surveillance" (CHIPS) retrospective and prospective observational cohort study by Italian Association of Pediatric Hematology and Oncology (AIEOP).

Background: Inherited thrombocytopenias (ITs) are rare congenital bleeding disorders characterized by different clinical expression and variable prognosis. ITs are poorly known by clinicians and often misdiagnosed with most common forms of thrombocytopenia. Material and methods: "CHildren with Inherited Platelet disorders Surveillance" study (CHIPS) is a retrospective - prospective observational cohort study conducted between January 2003 and January 2022 in 17 centers affiliated to the Italian Association of Pediatric Hematology and Oncology (AIEOP). The primary objective of this study was to collect clinical and laboratory data on Italian pediatric patients with inherited thrombocytopenias. Secondary objectives were to calculate prevalence of ITs in Italian pediatric population and to assess frequency and genotype-phenotype correlation of different types of mutations in our study cohort. Results: A total of 139 children, with ITs (82 male - 57 female) were enrolled. ITs prevalence in Italy ranged from 0.7 per 100,000 children during 2010 to 2 per 100,000 children during 2022. The median time between the onset of thrombocytopenia and the diagnosis of ITs was 1 years (range 0 - 18 years). A family history of thrombocytopenia has been reported in 90 patients (65%). Among 139 children with ITs, in 73 (53%) children almost one defective gene has been identified. In 61 patients a pathogenic mutation has been identified. Among them, 2 patients also carry a variant of uncertain significance (VUS), and 4 others harbour 2 VUS variants. VUS variants were identified in further 8 patients (6%), 4 of which carry more than one variant VUS. Three patients (2%) had a likely pathogenic variant while in 1 patient (1%) a variant was identified that was initially given an uncertain significance but was later classified as benign. In addition, in 17 patients the genetic diagnosis is not available, but their family history and clinical/laboratory features strongly suggest the presence of a specific genetic cause. In 49 children (35%) no genetic defect were identified. In ninetyseven patients (70%), thrombocytopenia was not associated with other clinically apparent disorders. However, 42 children (30%) had one or more additional clinical alterations. Conclusion: Our study provides a descriptive collection of ITs in the pediatric Italian population.

Prevalence and natural history of variants in the ANKRD26 gene: a short review and update of reported cases.

ANKRD26 is a highly conserved gene located on chromosome 10p12.1 which has shown to play a role in normal megakaryocyte differentiation. ANKRD26-related thrombocytopenia, or thrombocytopenia 2, is an inherited thrombocytopenia with mild bleeding diathesis resulting from point mutations the 5'UTR of the ANKRD26 gene. Point mutations in the 5'UTR region have been shown to prevent transcription factor-mediated downregulation of ANKRD26 in normal megakaryocyte differentiation. Patients with ANKRD26-related thrombocytopenia have a predisposition to developing hematological malignancies, with acute myeloid leukemia and myelodysplastic syndrome most commonly described in the literature. We review the clinical features and biological mechanisms of ANKRD26-related thrombocytopenia and summarize known cases in the literature.

Potential biomarkers for inherited thrombocytopenia 2 identified by plasma proteomics.

Inherited thrombocytopenia 2 (THC2) is difficult to diagnose due to the lack of specific clinical characteristics and diagnostic methods. To identify potential plasma protein biomarkers for THC2, we collected the plasma samples from a THC2 family (9 THC2 and 15 non-THC2 members), enriched the medium and low abundant proteins using Proteominer and analyzed the protein profiles using the liquid chromatography-mass spectrometry in data independent acquisition mode. Initially, we detected 784 proteins in the plasma samples of this family and identified 27 up-regulated and 36 down-regulated in the THC2 group compared to the non-THC2 group (|log2 ratio| >1 and p-value <0.05). To improve the predictive power, top eight dysregulated proteins (B7Z2B4, LTF, HP, ERN1, IGHV1-8, A0A0X9V9C4, VH6DJ, and D3JV41) were selected by an area under the curve-based random forest process to construct a clinical model. Multivariate analysis with random forest and support vector machine showed that the prediction model provided high discrimination ability for THC2 diagnosis (AUC: 1.000 and 0.967, respectively). The potential plasma protein biomarkers will be tested in more THC2 patients and other thrombocytopenia patients to further validate their specificity and sensitivity.

A Novel Mutation in GP1BB Reveals the Role of the Cytoplasmic Domain of GPIbß in the Pathophysiology of Bernard-Soulier Syndrome and GPIb-IX Complex Assembly.

Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbß, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbß. The loss of the intracytoplasmic tail of GPIbß results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbß; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbß is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.

Platelet features allow to differentiate immune thrombocytopenia from inherited thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder, for which no specific diagnostic test exists. Inherited thrombocytopenia (IT) can mimic ITP and lead to unappropriated management with significant morbidity. Here, in small cohorts of these two disorders, we explored whether platelet sialylation and platelet activation could allow to discriminate the two conditions. We also aimed to confirm the value of immature platelet counts in this discrimination. Platelet sialylation and the expression level of P-selectin were assessed by multiparameter flow cytometry. Immature platelets were estimated on a Sysmex XN 9000 analyzer. No significant difference in platelet sialylation was observed between ITP and IT. Contrarily, platelet activation was significantly higher in ITP patients (p = 0.008). The immature platelet fraction, as previously demonstrated, was significantly lower in the ITP group compared to the IT group (p = 0.014). That statistical significance was achieved in this small pilot study suggests that the two easily available assays of immature platelet count and P-selectin expression could help physicians to reach the proper diagnosis in complex cases of thrombocytopenia.

Miniaturized 3D bone marrow tissue model to assess response to Thrombopoietin-receptor agonists in patients.

Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias.

Platelets are tiny cell fragments essential for blood to clot. They are created and released into the bloodstream by megakaryocytes, giant cells that live in the bone marrow. In certain genetic diseases, such as Inherited Thrombocytopenia, the bone marrow fails to produce enough platelets: this leaves patients extremely susceptible to bruising, bleeding, and poor clotting after an injury or surgery. Certain patients with Inherited Thrombocytopenia respond well to treatments designed to boost platelet production, but others do not. Why these differences exist could be investigated by designing new test systems that recreate the form and function of bone marrow in the laboratory. However, it is challenging to build the complex and poorly understood bone marrow environment outside of the body. Here, Di Buduo et al. have developed an artificial three-dimensional miniature organ bioreactor system that recreates the key features of bone marrow. In this system, megakaryocytes were grown from patient blood samples, and hooked up to a tissue scaffold made of silk. The cells were able to grow as if they were in their normal environment, and they could shed platelets into an artificial bloodstream. After treating megakaryocytes with drugs to stimulate platelet production, Di Buduo et al. found that the number of platelets recovered from the bioreactor could accurately predict which patients would respond to these drugs in the clinic. This new test system enables researchers to predict how a patient will respond to treatment, and to tailor therapy options to each individual. This technology could also be used to test new drugs for Inherited Thrombocytopenias and other blood-related diseases; if scaled-up, it could also, one day, generate large quantities of lab-grown blood cells for transfusion.