Immune Thrombocytopenia Patient Research Articles

Identification of metabolism-related key genes as potential biomarkers for pathogenesis of immune thrombocytopenia

Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction. A biomarker is a biological entity that contributes to disease pathogenesis and reflects disease activity. Metabolic alterations are reported to be associated with the occurrence of various diseases. As metabolic biomarkers for ITP have not been identified. This study aimed to identify metabolism-related differentially expressed genes as potential biomarkers for pathogenesis of ITP using bioinformatic analyses.The microarray expression data of the peripheral blood mononuclear cells were downloaded from the Gene Expression Omnibus database (GSE112278 download link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112278). Key module genes were intersected with metabolism-related genes to obtain the metabolism-related key candidate genes. The hub genes were screened based on the degree function in the coytoscape sofware. The key ITP-related genes were subjected to functional enrichment analysis. Immune infiltration analysis was performed using a single-sample gene set enrichment analysis algorithm to evaluate the differential infiltration levels of immune cell types between ITP patient and control. Molecular subtypes were identified based on the expression of hub genes. The expression of hub genes in the ITP patients was validated using quantitative real-time polymerase chain reaction analysis. This study identified five hub genes (ADH4, CYP7A1, CYP1A2, CYP8B1, and NR1H4), which were be associated with the pathogenesis of ITP, and two molecular subtypes of ITP. Among these hub genes, CYP7A1 and CYP8B1 involved in cholesterol metabolism,were further verified in clinical samples.

Metabolomics profile and machine learning prediction of treatment responses in immune thrombocytopenia: A prospective cohort study.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and impaired platelet production. The mechanisms underlying ITP and biomarkers predicting the response of drug treatments are elusive. We performed a metabolomic profiling of bone marrow biopsy samples collected from ITP patients admission in a prospective study of the National Longitudinal Cohort of Hematological Diseases. Machine learning algorithms were conducted to discover novel biomarkers to predict ITP patient treatment responses. From the bone marrow biopsies of 91 ITP patients, we quantified a total of 4494 metabolites, including 1456 metabolites in the positive mode and 3038 metabolites in the negative mode. Metabolic patterns varied significantly between groups of newly diagnosed and chronic ITP, with a total of 876 differential metabolites involved in 181 unique metabolic pathways. Enrichment factors and p-values revealed the top metabolically enriched pathways to be sphingolipid metabolism, the sphingolipid signalling pathway, ubiquinone and other terpenoid-quinone biosynthesis, thiamine metabolism, tryptophan metabolism and cofactors biosynthesis, the phospholipase D signalling pathway and the phosphatidylinositol signalling system. Based on patient responses to five treatment options, we screened several metabolites using the Boruta algorithm and ranked their importance using the random forest algorithm. Lipids and their metabolism, including long-chain fatty acids, oxidized lipids, glycerophospholipids, phosphatidylcholine and phosphatidylethanolamine biosynthesis, helped differentiate drug treatment responses. In conclusion, this study revealed metabolic alterations associated with ITP in bone marrow supernatants and a potential biomarker predicting the response to ITP.

Evaluation of ISTH Severe Bleeding Criteria and Development of a Mortality Prognosis Model for Hospitalized Immune Thrombocytopenia Patients: A Multicenter, Retrospective and Prospective Cohort Study

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by the immune system's erroneous destruction of platelets, which play a crucial role in blood clot formation. This condition precipitates a decreased platelet count and consequently heightens the risk of bleeding events, ranging from mild petechiae to severe, potentially fatal occurrences such as intracranial hemorrhage. Therefore, accurate anticipation and management of these severe bleeding risks form an integral facet of clinical care for ITP patients. The International Society on Thrombosis and Hemostasis (ISTH) has instituted criteria designed to classify and forecast severe bleeding in patients with thrombotic disorders. Despite their widespread use in diverse clinical scenarios, the validity and efficacy of these criteria for ITP patients remain underexplored. Furthermore, disparities in the application and interpretation of these criteria among practitioners underscore the necessity for a comprehensive evaluation within the ITP context. Consequently, our study is poised to fill these crucial gaps. We intend to evaluate the performance and applicability of the ISTH severe bleeding criteria specifically within the ITP patient population. Concurrently, we aspire to develop and validate a robust, reliable mortality prediction model for this unique group of hospitalized patients. Methods: This study was a multicenter, retrospective cohort investigation conducted across 20 medical centers nationwide. A total of 1487 adult patients diagnosed with immune thrombocytopenia were enrolled. The study period spanned from Jan 2020 to Jan 2023, ensuring a comprehensive assessment of patient outcomes. To further evaluate the joint impact of multiple prognostic factors, a multivariate Cox regression model was constructed. Prospective observational cohorts were subsequently established with the objective of assessing the real-world performance of the mortality prognosis model. Results: In our study, out of the 1487 enrolled adult patients with immune thrombocytopenia (ITP), 420 patients (28.24%) experienced bleeding events. The incidence of major bleeding types among these patients was assessed based on the critical bleeding diagnostic criteria. Intracranial bleeding was found to be the most common type, occurring in 229 patients (54.46%). This was followed by respiratory compromise in 87 patients (20.66%) and hemodynamic instability bleeding in 76 patients (18.08%). Intramuscular bleeding was the least common, observed in only 3 patients (0.70%). Furthermore, we evaluated the efficacy of the ISTH severe bleeding criteria in predicting mortality among ITP patients. Our analysis unveiled a significant association between meeting the ISTH severe bleeding criteria and increased mortality rates. Patients who met the criteria demonstrated a higher incidence of death compared to those who did not meet the criteria. Additionally, we developed a mortality prognosis model for hospitalized ITP patients, incorporating various clinical and laboratory parameters. The model considered the presence of intracranial bleeding, admission platelet count, cardiovascular disease, diabetes mellitus, and admission creatinine levels as predictive factors. The mortality prognosis model demonstrated strong predictive power, with an area under the receiver operating characteristic curve of 0.82. This indicates its ability to distinguish between patients at high and low risk of mortality. Kaplan‒Meier survival analyses further confirmed significant survival differences between patients who met the ISTH severe bleeding criteria and those who did not. Conclusions: Our study reaffirms the substantial relevance of ISTH severe bleeding criteria in assessing bleeding profiles and prognosis among hospitalized ITP patients. The developed mortality prognosis model demonstrates excellent predictive capabilities for mortality in this patient population and is currently undergoing validation in a prospective cohort. These findings emphasize the importance of individualized management strategies to optimize outcomes in ITP patients. Further research and validation are warranted to refine and validate the mortality prognosis model for broader clinical application.

Real-World Treatment Patterns and Outcomes in Patients with Immune Thrombocytopenia Treated with Avatrombopag in the United States: Real-AVA 2.0 Study Design

Background: Thrombopoietin-receptor agonists (TPO-RAs) are used in the treatment of chronic immune thrombocytopenia (ITP), a disorder characterized by prolonged low platelet counts (PCs) that pose a risk of serious bleeding events. The TPO-RAs eltrombopag (ELT) and romiplostim (ROMI) have been available for more than 10 years. ELT is an oral medication with food restrictions and carries a boxed warning for hepatoxicity, requiring hepatic laboratory monitoring and a potential need for statin dosing adjustments. ROMI, an injectable, is typically administered in a health care practitioner's office on a weekly basis without food restrictions. Avatrombopag (AVA), an oral medication taken with food, is the most recently approved TPO-RA for the treatment of chronic ITP (2019 by FDA and 2021 by EMA). Hence, the real-world evidence on AVA treatment and its clinical effectiveness in broader and more diverse ITP patient populations have been limited, and additional studies are warranted. REAL-AVA 2.0 is a retrospective chart review which will gather a substantially greater amount of platelet counts per patient than either a claims database review or an electronic medical record review. More granular efficacy data with more patients, more platelet counts per patient, and in a broader population will be used to better assess the clinical effectiveness of AVA in the real-world. This study will also describe the demographic and clinical characteristics of adult patients with primary ITP treated with AVA in the United States (US), and assess treatment patterns and clinical outcomes of these patients. Methods: This retrospective, observational medical chart review study will include patients treated in the US with AVA in a non-clinical trial setting from approximately 20 centers (both academic and community). The study aims to include approximately 200 ITP patients (at least 25 with an ITP disease duration < 12 months) treated with at least one dose of AVA between July 2019 and March 2023. The date the patient initiated treatment with AVA is defined as the index date. The period from the index date through the study end date, end of data availability for the patient or date of death, whichever occurs first, is defined as the post-index period. Results: The primary objective of the study is to describe treatment patterns and response to AVA. Primary outcome measures to be assessed include: achievement of meaningful PC thresholds (PC ≥ 30 X10 9/µL and ≥ 50 X10 9/µL) at any time during AVA treatment, time to achieve an initial platelet response with AVA based on achieving meaningful PC thresholds (PC ≥ 30X10 9/µL and ≥ 50 X10 9/µL) after AVA index date and before AVA discontinuation, durability of platelet response among patients who respond to AVA, and reduction in the use of concomitant steroids or immunosuppressant agents from the index date. The secondary objectives include: assessment of the demographic and clinical characteristics of patients treated with AVA, assessment of changes in PCs following the index date, ITP-related medication use prior to and following the index date, and treatment patterns with AVA (including treatment persistence and changes in dose/frequency). The exploratory objectives include assessment of AVA effectiveness in specific subpopulations (e.g. prior number of ITP treatments, persistent vs chronic ITP, switch from particular ITP treatments, gender, race, age) and, if feasible, to evaluate social determinants of health among ITP patients treated with AVA (including assessment of treatment response among those subpopulations of interest) [ Table 1]. Summary/Conclusions: The study aims to fill current gaps in knowledge by assessing treatment patterns and outcomes, clinical, and demographic characteristics of patients treated with AVA in a real-world clinical setting in the US. We look forward to presenting this data at upcoming scientific meetings.

Clinical and immunological characterisation of patients with common variable immunodeficiency related immune thrombocytopenia

Primary Immune thrombocytopenia (ITP) is an autoimmune disease. Secondary ITP occurs in patients with underlying diseases such as common variable immunodeficiency (CVID). CVID is one of the most common symptomatic primary immunodeficiencies in adults, characterised by infectious and non-infectious symptoms. Amongst CVID patients, ITP is the most frequent autoimmune manifestation. In this single-centre study, we performed a clinical and immunological characterisation of 20 patients with CVID-related ITP and 20 ITP patients without CVID to compare severity and remission rates. We found that patients with CVID-related ITP had a higher WHO Bleeding Scale at initial diagnosis yet showed higher remission rates and required less treatment. Patients with ITP needed up to seven therapy options and were often treated with second-line drug therapy, whilst only one CVID-related ITP patient required second-line drug therapy. Therefore, we show that the course of thrombocytopenia in patients with CVID-related ITP is milder. Furthermore, we show that soluble interleukin-2 receptor (sIL-2R, CD25) was higher in CVID-related ITP compared to ITP patients and could accurately classify patient cohorts with an Area Under the Receiver Operating Characteristic of 0.92. Whilst none of the ITP patients had a history of immunodeficiency, we found immunological abnormalities in 12 out of 18 patients. Therefore, we recommend screening ITP patients for CVID and other immunodeficiencies to detect immune abnormalities early, as we found patients with reduced immunoglobulin levels as well as severe lymphocytopenia in our ITP cohort.

Discontinuation of Thrombopoietin Receptor Agonists: A 10 Year Real-World Experience from an Academic Hospital in Madrid

Background: Thrombopoietin receptor agonists (TPO-RAs) have become the second line treatment for patients with immune thrombocytopenia (ITP). Despite TPO-RAs are currently indicated for continuous treatment, some patients can achieve a sustained response that allows tapering and discontinuation. Predictors of responses and relapses after discontinuation are unknown factors that are being under investigation. Methods: This is a retrospective study approved by Hospital Universitario La Paz Ethical Committee. We describe our experience during 10 years in discontinuation of TPO-RAs and relapses in the ITP patient cohort from Hospital Universitario La Paz in Madrid.We included all ITP patients treated with TPO-RAs between 1st January 2011 and 31st December 2020. We analyzed response, discontinuation, and relapses after TPO-RAs treatments, with a follow-up period until 31st December 2021. This research was funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association. Results: One-hundred-fifty-one ITP patients were treated with TPO-RAs between 1st January 2011 and 31st December 2020. We excluded those without complete information about response, discontinuation, and relapses after TPO-RAs, analyzing a total of 133 patients. Thirteen received Romiplostim, 7 men and 6 women, with a median age of 66y (range 3-98). All of them achieved a clinical response according to standard criteria and in 4 it was possible to discontinue TPO-AR, with no need of retreatment with a median follow-up of 17,4 months (13,6-39,5). Seventy-five patients were treated with Eltrombopag, 33 men and 42 women, with a median age of 49y (range 1-98). Seventy-one patients get a clinical response, which allows discontinuation of TPO-AR in 43 of them, with 25 sustained responses and a median follow-up of 40,5 months (9,9-60,3). We also included 45 ITP patients, 21 men and 24 women, median age of 53y (range 2-93), who received both TPO-AR because of lack of response or intolerance with the first (4 Romiplostim and 41 Eltrombopag). Among the 34 patients who achieved a response, 17 were able to discontinue TPO-ARs and 7 needed retreatments. The follow-up period for this cohort was 23,4 months (1,9-75). Globally, 39 patients discontinue TPO-RAs with no need of further ITP treatment with a median follow-up of 28,4months (1,9-75) since discontinuation until the end of the study.Results are summarized in Table 1. Conclusion: In conclusion, 29% (39/113) of ITP patients who were treated with TPO-RAs could discontinue this treatment without relapses with a median follow-up of 28,4months (1,9-75) since discontinuation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating ITP Plasma for Effect on Platelet Production Using the Autologous Peripheral Blood Based Megakaryopoiesis Assay

Investigating ITP plasma for effect on platelet production using the autologous peripheral blood based megakaryopoiesis assay Nikola Ivetic, Ishac Nazy, Rumi Claire, Angela Huynh John G. Kelton, Donald M. Arnold. Background: Studies focused on megakaryocyte number and function are needed to understand platelet underproduction as a potential mechanism of immune thrombocytopenia (ITP). Most experimental studies have relied on animal models, specialized cell lines, or exogenous and non-autologous sources of hematopoietic stem and progenitor cells (HSPCs) as a source of megakaryocytic cells and platelet production. We used HSPCs isolated from ITP patients themselves to examine the effects their plasma has megakaryocyte growth and platelet production. Study Design and Methods: Megakaryocytes were produced from peripheral blood CD34+ HSCs directly from patients with ITP and healthy controls. HSPC (CD45+ CD34+) were isolated, expanded for 4 days and then cultured with thrombopoietin (TPO) and stem cell factor (SCF) for 8 days. Megakaryocyte numbers were evaluated by flow cytometry using CD41a expression for megakaryopoiesis and CD42b expression for maturation. Platelet production was analyzed using CD41a expression and platelet forward and side scatter profile sizes. The addition of autologous ITP plasma and plasma depleted of IgG were also tested to determine the effects of antibodies on megakaryopoiesis and thrombopoiesis. Results: Peripheral blood derived megakaryocytes were cultured from HSPCs from ITP patients (N=10), and normal controls (N=9). Approximately 2,200±1,100 hematopoietic stem and progenitor cells were isolated per ml of peripheral blood and a 5-fold increase in stem cells was obtained after an expansion of the isolated ITP patient HSPC population. Following an additional 8 days of culture with TPO, 67±12% of the cultured cells were CD41a+ indicating they were undergoing megakaryopoiesis and 54±15% of the CD41a+ population expressed the CD42b marker for mature megakaryocytes. Of the 10 ITP cultures grown, four grew significantly more mature megakaryocytes in comparison to healthy controls. The addition of ITP plasma did not inhibit megakaryocyte growth or maturity, but platelet production was reduced with two samples demonstrating a 50% reduction in platelet levels in comparison to the . Relative to the number of mature megakaryocytes cultured there was a lower number of platelets detected in ITP cultures (3.34±1.9 platelets per mature megakaryocyte) in comparison to the healthy control group (8.1±1.4 platelets per mature megakaryocyte). Conclusions: The addition of patient plasma had no inhibiting effect on megakaryocyte counts in an autologous assay but a significant decrease in platelet number (thrombopoiesis) was detected. A subset of ITP HSPC generated more megakaryocytes when compared to healthy donors, indicating these cells are generating more megakaryocytes. This is to our knowledge the first autologous investigation of ITP plasma on patient HSPC undergoing megakaryopoiesis.

TCR CDR3 Sequencing as a Clue to Elucidate the Landscape of Dysimmunity in Patients with Primary Immune Thrombocytopenia.

Background: Primary immune thrombocytopenia (ITP) is an autoimmune disorder. The existence of autoreactive T cells has long been proposed in ITP. Yet the identification of autoreactive T cells has not been achieved, which is an important step to elucidate the pathogenesis of ITP. Methods: ITP patients’ peripheral blood was collected prior to the treatment and one month after initiating dexamethasone treatment per related therapeutic guideline. Serum cytokines were profiled to examine T cell subtypes imbalance using a protein chip. TCR Vβ analysis in CD8+T cells of ITP patients, and TCR CDR3 DNA sequencing of CD4+T and CD8+T cells were performed to determine the autoreactive T cells’ clones. Results: Cytokine profiling revealed imbalanced distribution of T cells subtypes, which was confirmed by CD4+T and CD8+T cells’ oligoclonal expansion of TCR Vβ analysis and TCR CDR3 DNA sequencing. VDJ segments were found to be more frequently presented in ITP patients, when compared with health controls. There was an individualized CD4+T cell or CD8+T cell CDR3 sequence in each ITP patient. Conclusions: The present study revealed that T cell clones expanded in ITP patients’ peripheral blood, and each clone had an individualized TCR CDR3 sequence. The expanded T cell clones preferred to use some specific VDJ segment. Further studies are warranted to get access to individualized treatment such as Car-T in patients with ITP.

Expansion of Human Megakaryocyte-Lineage Progeny via Aryl Hydrocarbon Receptor Antagonism with CH223191.

Aryl hydrocarbon receptor (AhR) antagonism is known to expand human hematopoietic stem cells (HSCs). However, its regulatory effect on the lineage-skewed differentiation of HSCs has not been sufficiently studied. Here, we investigate the effect of the AhR-selective antagonist CH223191 on the regulation of HSC differentiation. Consistent with the well-known effects of AhR antagonists, CH223191 treatment increase phenotypic HSCs (Lin-CD34 + CD38-CD90 + CD45RA-) and preserves their functionality. On the other hand, CH223191 leads to an overall expansion of megakaryocyte (MK)-lineage populations, such as MK progenitors (MKps, CD34 + CD41 +), immature MKs (CD41 + CD42b-), and mature MKs (CD41 + CD42b +), and it also activates MK/platelet-associated signaling pathways. Furthermore, CH223191 expands MKps, mature MKs, and p-selectin (CD62p)-positive platelet-like particles in immune thrombocytopenia (ITP) patient bone marrow (BM). These results highlight the numerical expansion of human MK-lineage progeny through AhR antagonism with CH223191. This approach using CH2231291 may be applicable in the development of auxiliary treatment regimens for patients with abnormal thrombopoiesis.

Successful phytotherapy for a patient with severe immune thrombocytopenia and failed with corticosteroids, azathioprine, eltrombopag, and platelet transfusion – A case report

The purpose of this study was to investigate and evaluate the effectiveness of phytotherapy on a severe and complicated Immune Thrombocytopenia (ITP) patient who had failed with conventional treatments. A male patient presented with clinical symptoms of ITP and had been treated with Corticosteroids, Azathioprine, Eltrombopag, and platelet transfusions for over three years. The patient had an initial response but later developed severe complications, including hydrothorax, gastric pain, hematuria, and digestive hemorrhage, and no further response to treatment. The patient then received Phytotherapy for 17 months which significantly improved the clinical symptoms, platelet counts, and laboratory tests. Despite his active lifestyle, the patient was symptom-free with platelet counts ranging from 109 to 132×109/L.

Adapted guideline for the diagnosis and treatment of primary immune thrombocytopenia for Chinese children (2021)

Adapted guideline for the diagnosis and treatment of primary immune thrombocytopenia for Chinese children (2021)

Do Splenectomized Immune Thrombocytopenia (ITP) Patients Have Increased Risks for Platelet Decreases Following COVID-19 Vaccination?

Do Splenectomized Immune Thrombocytopenia (ITP) Patients Have Increased Risks for Platelet Decreases Following COVID-19 Vaccination?

Efficiency and Safety of Eltrombopag for Multi-Line Failed Chinese Patients with Immune Thrombocytopenia: Cases with Decreased Megakaryocyte Response Well from Single Center Experience

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by decreased platelet (PLT) count in peripheral blood and marrow megakaryocyte dysfunction, which was induced by antiplatelet antibodies or cytotoxic T cells. Many ITP patient were previously full response to first-line treatment such as corticoids (CIS) or immunoglobulin (IVIG), but were intolerant or relapse during the maintenance treatment, and turn to receive recombinant human thrombopoietin (rhTPO), rituximab (RTX), splenectomy as well as immunosuppressive therapy (IST). Unfortunately, there still some patients failed to multiple treatments are faced with high bleeding risk.Eltrombopag, the first oral non-peptide thrombopoietin receptor agonist (TPO-RA) that approved by US FDA in 2008, is the second-line treatment option for chronic ITP based on guideline recommendation. As reported, the effective rate of eltrombopag in ITP ranges from 59% to 89.7%, and limited predictive factors were available to assess the efficiency for individual. The characteristic of marrow megakaryocyte (MK) in ITP was reported as normal or hyper megakaryocytosis on the initiation (before treatment), however there are also ITP patients with decreased marrow MK, and the response of eltrombopag to these patients have not been fully reported. Bone marrow aspiration is a routine examination in China for the diagnosis of ITP with severe thrombocytopenia. Therefore, we conducted a research tried to assess the efficiency of eltrombopag to patients with multi-line failed ITP, and analyzing the possible factors may contribute to the differences based on personal characteristic.Thirty-five multi-line failed ITP patients with PLT count bellow 30× 10 9/L who received eltrombopag treatment were enrolled, and divided into three groups, the hyper-MK (MK count>35, n=17), normal-MK (8-35, n=10) and hypo-MK (≤7, n=8) groups. The average PLT count was 10.63±6.30×10 9/L before eltrombopag treatment, and it increased significantly to 12 (2-65)×10 9/L, 39 (2-212)×10 9/L, 68 (2-453)×10 9/L, 60 (3-358)×10 9/L, and 75 (3-308)×10 9/L in the 1st, 2nd, 4th, 6th , and 8th weeks after treatment (all P=0.000, except 1st week). The median intervals time required for platelet to reach 30×10 9/L and 100×10 9/L for the first time were 11 (4-40) and 21 (10-65) days, respectively (Fig. A-C). We found the overall, complete (PLT count ≥100×10 9/L) and partial response (PLT count ≥30×10 9/L or at least twice the baseline value) rates were 54.3% (n=19), 48.6% (n=17), and 5.7% (n=2) respectively to eltrombopag in our center. The overall response rate of patient with decreased MK was 75%, which was unexpectedly higher than the patient with increased or normal MK count (52.9% and 40%, respectively) (Fig. D). To explain the underling mechanism, we detected the peripheral T lymphocyte, B lymphocytes and NK cells before eltrombopag. Results showed that the patient with decreased MK were characteristic with higher T helper (Th) cells (39.62±2.01%) and regulatory T (Tregs) cells (7.93±1.63%) when compared to the hyper-MK (30.44±10.95%, 4.23±1.67%) and normal-MK group (25.67±5.72%, 4.81±2.12%). For patient with poorer eltrombopag response group, more percentage and absolute number of NK cells (15.48±7.12%, 234.4±91.80×10 6/L) were found in peripheral blood (Fig. E-F). We also discovered that previous first-line (CIS or IVIG) treatment, rhTPO effectiveness, RTX splenectomy and IST had no influence on eltrombopag response (Fig. G). As for safety, nine eltrombopag related adverse events were reported, and most commonly were upper respiratory tract infection (8.6%), elevated ALT (5.7%), and venous thrombosis (5.7%). All the side effect was cured by symptomatic treatment, eltrombopag dose reduction or discontinue.In summary, ITP patients with decreased megakaryocyte respond well to eltrombopag, and the abnormality of NK cells may play a role in patients exert a poor response. In further clinical practice, we should considering the megakaryocytes count as well as peripheral NK population to better predicting eltrombopag response in ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (<100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa.Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy.Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane.The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%).The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts. DisclosuresPatel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.

Contribution of Next Generation Flow (NGF) Cytometry in Primary Immune Thrombocytopenia (ITP): Utility for the Differential Diagnosis with Myelodysplastic Syndromes

Background: Primary immune thrombocytopenia (ITP) is an immune-mediated acquired disorder characterized by impaired production and increased destruction of platelets with an elevated risk of bleeding. At present, diagnosis of primary ITP still remains one of exclusion with a need to discard other causes of isolated thrombocytopenia, in the absence of robust and accurate clinical and laboratory diagnostic criteria. For the diagnosis of ITP, a bone marrow (BM) study may be useful to differentiate between ITP and other diseases, such as myelodysplastic syndromes (MDS). Next generation flow (NGF) has emerged as a potential useful tool in these settings.Aim: In this study (FCR-PTI-2017-01) we prospectively evaluated the potential utility of NGF analysis of BM and peripheral blood (PB) for more accurate diagnosis of ITP vs MDS.Methods: 62 patients presenting with isolated thrombocytopenia and classified as ITP (n=20), MDS (n= 11) or inconclusive, i.e. unclassifiable, (n= 25) by BM cytomorphology, were studied. PB (n=47) and BM (n=62) analysis by NGF was blindly performed in parallel for the ITP, MDS and unclassifiable patient groups using the EuroFlow 8-colour AML/MDS classification antibody panel followed by automated analysis against pre-existing flow cytometry databases of normal healthy donor PB and BM immunophenotypic profiles. NGF BM and PB results were then compared with the BM cytomorphological diagnosis. In parallel, epidemiological data from patients were recorded in an electronic case report form (eCRF) and analyzed afterwards.Results: By cytomorphology, expert hematologists were able to conclude an ITP or MDS diagnosis in only 31 cases (55.3%). 62 BM and 48 PB samples with isolated thrombocytopenia were evaluated by NGF. Our 62 patients were allocated in 4 immunophenotypic groups attending to different BM variables observed: maturation blockades, abnormal antigen expression and cross lineage markers. Thus, we observed normal phenotype cases (n=10), isolated (unilineage) alterations (n=24), mild multilineage (>1) alterations (n=20) and severe multilineage (MDS-like) phenotypes (n=8). Cytomorphology diagnosed our cases as ITP, MDS or unclassifiable with a median number of alterations observed by BM NGF of 4 (IQR, 3-6), 2 (IQR, 1-6) and 4 (IQR, 3-5) respectively. For ITP cytomorphology group, NGF demonstrated numerous BM alterations being monocytic alterations (n=17, 94%) the most frequent finding observed. MDS presumed cases were also associated with monocytic alterations (50%) with a frequent decrease in neutrophil precursors (40%). On the contrary, when cytomorphology was not capable to establish a diagnosis, NGF showed a mixture of alterations with no clear predominance of none of them (table 1). Similarly to our work with BM NGF, we looked into a potential correlation of cytomorphology with PB NGF phenotypes. Thus, we observed a median number of alterations of 4 (IQR, 3-5), 4 (IQR, 2-4) and 4 (IQR, 3-5) in ITP, MDS and inconclusive cytomorphology groups. Increased platelet size and upregulated CD41, CD61 and CD63 glycoprotein (GP) expression were the most characteristic findings of ITP cohort. MDS subtype depicted an increased platelet size and overexpression of CD41. Downregulation of GP was restricted to patients with MDS-like phenotypes. (table 2). Nearly statistical significant differences at significance level of 90% were observed between cytomorphology and BM NGF results (p=0.179) (table 3), between platelet score and BM NGF findings (p=0.118) and also, among morphology, BM NGF and platelet score (p=0.179). Nevertheless, cytomorphology and PB NGF showed no statistical differences between them (p=0.206) (table 4).Conclusions: Limitations of BM cytomorphology when facing an isolated thrombocytopenia were demonstrated here. However, normal or unilineage BM NGF alterations may lead us to an ITP diagnosis while mild or severe multilineage BM phenotypes may correlate good with MDS. PB platelet GP expression allowed us to classify our patients in six groups (Gonzalez-Lopez/Matarraz platelet score) which may help ITP diagnosis when this score is low. Comparison of BM cytomorphology, BM NGF and PB NGF techniques at diagnosis showed statistically nearly comparable results (p=0.179), with a bigger amount of patients needed to confirm this trend. All these NGF findings may lead us to better address ITP at diagnosis. [Display omitted] DisclosuresGonzalez-Lopez: Novartis: Other: Advisoryboard and speakers honoraria, Research Funding; Amgen: Other: Advisory board and speakers honoraria, Research Funding; Sobi: Other: Advisory board honoraria; Grifols: Other: Advisory board honoraria.

Abnormalities of bone marrow B cells and plasma cells in primary immune thrombocytopenia

AbstractPrimary immune thrombocytopenia (ITP) is an autoantibody-mediated hemorrhagic disorder in which B cells play an essential role. Previous studies have focused on peripheral blood (PB), but B cells in bone marrow (BM) have not been well characterized. We aimed to explore the profile of B-cell subsets and their cytokine environments in the BM of patients with ITP to further clarify the pathogenesis of the disease. B-cell subpopulations and their cytokine/chemokine receptors were detected by using flow cytometry. Plasma concentrations of cytokines/chemokines were measured by using enzyme-linked immunosorbent assay. Messenger RNA levels of B cell–related transcription factors were determined by using quantitative polymerase chain reaction. Regulatory B cell (Breg) function was assessed by quantifying their inhibitory effects on monocytes and T cells in vitro. Decreased proportions of total B cells, naive B cells, and defective Bregs were observed in patients with ITP compared with healthy controls (HCs), whereas an elevated frequency of long-lived plasma cells was found in BM of autoantibody-positive patients. No statistical difference was observed in plasmablasts or in short-lived plasma cells between patients with ITP and HCs. The immunosuppressive capacity of BM Bregs from patients with ITP was considerably weaker than HCs. An in vivo study using an active ITP murine model revealed that Breg transfusion could significantly alleviate thrombocytopenia. Moreover, overactivation of CXCL13-CXCR5 and BAFF/APRIL systems were found in ITP patient BM. Taken together, B-cell subsets in BM were skewed toward a proinflammatory profile in patients with ITP, suggesting the involvement of dysregulated BM B cells in the development of the disease.

Autoimmune Cytopenias (AIC) Following Allogeneic Haematopoietic Stem Cell Transplant for Acquired Aplastic Anaemia: A Joint Study of the Autoimmune Diseases and Severe Aplastic Anaemia Working Parties (ADWP/SAAWP) of the European Society for Blood and Marrow Transplantation (EBMT)

IntroductionAutoimmune cytopenias(AICs) are the most common autoimmune phenomenon encountered following haematopoietic stem cell transplant(HSCT) (Holbro et al. 2012). Mechanisms of autoimmunity post-HSCT may relate to genetic susceptibility(Consolini et al. 2016), failure of central and peripheral immune tolerance(King et al. 2004), and factors such as drugs and inflammation(Lang et al. 2005). The aim of this EBMT study is to describe the incidence, risk factors, diagnosis and treatment of AIC post alloHSCT for acquired aplastic anaemia (aAA), itself a recognized autoimmune disease in most patients.MethodsEBMT centres performing alloHSCT for aAA were invited to participate in the study. Participating centres identified paediatric and adult patients with aAA treated between January 2002 and December 2012 with first alloHSCT, and confirmed whether AIC was diagnosed post-HSCT. Patients diagnosed with AIC prior to HSCT were excluded. Data for patients without AIC were extracted from the EBMT registry. Centres identifying cases of AIC provided additional data on diagnostic criteria and investigations, therapy administered and treatment outcome.Results196 (38%) paediatric and 320 (62%) adult patients were confirmed eligible for study inclusion by 40 participating EBMT centres. Characteristics of n=516 patients are shown in Table 1. 23 patients were diagnosed at a median of 13.0 (range 2.6-91.4) months post alloHSCT with AICs as follows: 30.4% (n=7) immune thrombocytopenia (ITP), 26.1% (n=6) autoimmune haemolytic anaemia(AIHA), 17.4% (n=4) autoimmune immune neutropenia (AIN), and 21.7% (n=5) Evans Syndrome. 1 case (4.3%) was diagnosed both with AIHA and AIN. Cumulative incidence of AIC at 1, 3 and 5 years post AIC was 2.0% (95% CI 0.7-3.2%), 4.1% (2.3-5.9%) and 4.4% (2.6-6.3%) respectively. Cumulative incidence of AIC at 5 years post HSCT did not vary with patient age group (<18 vs ≥18), Interval from aAA diagnosis to transplant (<12 vs ≥12 months), donor/recipient CMV match, donor/recipient gender match, or conditioning with TBI (P>0.5 in all cases). However, 5-year cumulative incidence of AIC was higher amongst patients treated with non-myeloablative conditioning (6.9% vs 2.5%, p=0.03), and conditioning regimens containing Fludarabine (6.2 vs 1.9%, p=0.03) or Alemtuzumab (10.0% vs 2.93%, p=0.003). Conditioning with anti-thymocyte globulin(ATG) was associated with a lower incidence of AIC (1.6 vs 6.2%, p=0.02). Incidence of AIC was also significantly higher in patients receiving PBSC (9.9%) compared with UCB (5.0%) and BM (0.8%) (p=0.002). 2 AIC patients received a second HSCT, one 2.3 months after AIC diagnosis and one 35 months after AIC diagnosis. There were 3 deaths in the AIC group, 2 infection-related to AIC and 1 HSCT-related.Diagnostic investigations varied across centres with direct antiglobulin test (DAT) performed in 6/7 AIHA patients, and bone marrow examination (BME) performed in 5/7 ITP patients. Anti-neutrophil antibodies were screened for in all AIN cases and detected in 1 patient. Multiple lines of therapy were required for patients with ITP, AIHA and Evans syndrome with 42.1%(n=8) of these patients not responding, and a further 26.3% (n=5) relapsing after initial complete response (CR) to first-line therapy. 1 ITP patient exhibited disease refractory to 3 lines of therapy including splenectomy. 2 AIHA patients achieved CR, and 1 patient with Evans syndrome achieved partial response following 4 lines of therapy. 2 AIN patients did not require treatment; 1 AIN patient achieved PR after first line treatment with granulocyte-colony stimulating factor (G-CSF), and a second achieved CR after second-line treatment with G-CSF and Rituximab.ConclusionsWith a cumulative incidence of 4.4% (2.6-6.3%) over 5 years, AICs are a clinically challenging and potentially life-threatening complication of alloHSCT for aAA. Patients receiving non-myeloablative conditioning regimens incorporating Fludarabine and/or Alemtuzumab, and those receiving PBSC are at higher risk of this complication. Diagnosis of AIC should be considered and investigated in cases of new or worsening cytopenia at any stage post alloHSCT for aAA. Standardization of diagnostic and therapeutic approaches, along with data registration (via the EBMT and other databases), is warranted to inform harmonized recommendations for management. [Display omitted] DisclosuresSnowden:Sanofi: Honoraria. Bader:Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding. Peffault De Latour:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.

The Treatment Options and Clinical Significance of Immune Thrombocytopenia Patients with Splanchnic Vein Thrombosis as the Initial Manifestation

To investigate the causes, treatment options and outcomes of immune thrombocytopenia (ITP) patients with splanchnic venous thrombosis (SVT). The clinical diagnosis, treatment and outcomes data of one 26-year-old male ITP patient with SVT as initial manifestation were collected. The possible causes and treatment options of the patients were discussed through literatures review. The result of blood routine tests of the patient showed that Plt（17-38）×109/L and eosinophils (EOS) 46.9%-50.0%. B-ultrasound and CT findings suggested thromboses of splenic vein, hepatic portal vein and its branches were formed, the patient was diagnosed as ITP with SVT. After active etiological treatment and reduced dose of low molecular weight heparin(LMWH) for anticoagulation, the patient recovered. ITP combined with large scale of SVT is rare, and it is difficult to cure. It should be pay more attention to the possible thrombosis risk triggered by a transiently increased EOS in the blood stream. Promptly etiological treatment and the balance between anticoagulant therapy and bleeding risks should be taken in clinical practice.

Platelet function and bleeding at different phases of childhood immune thrombocytopenia

Immune thrombocytopenia (ITP) is believed to be associated with platelet function defects. However, their mechanisms are poorly understood, in particular with regard to differences between ITP phases, patient age, and therapy. We investigated platelet function and bleeding in children with either persistent or chronic ITP, with or without romiplostim therapy. The study included 151 children with ITP, of whom 56 had disease duration less than 12 months (grouped together as acute/persistent) and 95 were chronic. Samples of 57 healthy children were used as controls, while 5 patients with leukemia, 5 with aplastic anemia, 4 with MYH9-associated thrombocytopenia, and 7 with Wiskott-Aldrich syndrome were used as non-ITP thrombocytopenia controls. Whole blood flow cytometry revealed that platelets in both acute/persistent and chronic ITP were increased in size compared with healthy donors. They were also pre-activated as assessed by PAC1, CD62p, cytosolic calcium, and procoagulant platelet levels. This pattern was not observed in other childhood thrombocytopenias. Pre-activation by CD62p was higher in the bleeding group in the chronic ITP cohort only. Romiplostim treatment decreased size and pre-activation of the patient platelets, but not calcium. Our data suggest that increased size, pre-activation, and cytosolic calcium are common for all ITP platelets, but their association with bleeding could depend on the disease phase.

I-WISh: A wish list for immune thrombocytopenia quality of life indicators becomes reality.

I-WISh: A wish list for immune thrombocytopenia quality of life indicators becomes reality.