Abstract: The diagnosis and management of thrombocytopenia is a growing component in the practice of hematology. The frequency with which hematologists are called in consultation for thrombocytopenia continues to increase with the advent of routine automated platelet determinations and the introduction of new medications. For most patients, such as those with inherited and auto-immune thrombocytopenia, emphasis is focused on efforts to treat or forestall bleeding without excess drug-induced toxicity or burden to the patient. However, in disorders such as heparin-induced thrombocytopenia (HIT), avoidance of thrombotic complications is the key to management. In this chapter, we provide the pediatric and adult hematologist with new insights into the pathogenesis and recognition of congenital inherited thrombocytopenias (CTP), a hitherto difficult to comprehend constellation of clinical entities. We also highlight new approaches to the diagnosis and treatment of two of the more common thrombocytopenic conditions encountered in practice, autoimmune or idiopathic thrombocytopenic purpura (ITP) and HIT. In Section I, Dr. James Bussel discusses CTPs and their distinction from childhood ITP. He emphasizes the clinical features that enable the pediatrician and hematologist to suspect the diagnosis of CTP and those that are of use to subcategorize the various entities, where possible. He also emphasizes newer molecular markers that afford definitive diagnosis in some cases and provide insight into platelet production. This section highlights the characteristic associated findings and differences in the natural history and approaches to management of the various entities. In Section II, Dr. Robert McMillan discusses adult chronic ITP. He revisits the utility of platelet antibody determination in diagnosis and review new insights into pathogenesis. The role of Helicobacter pylori infection and the timing of splenectomy in the management of acute and emergent ITP are examined. New insights into the natural history of ITP post-splenectomy and management strategies for patients with severe, chronic, refractory ITP are discussed. In Section III, Dr. James Zehnder updates us on HIT. He emphasizes new insights into the clinical presentation and pathogenesis of this condition. He critically reviews the utility of laboratory testing for heparin-dependent antibodies. Recent studies on the use of direct thrombin inhibitors are examined and the management of cardiopulmonary bypass surgery in patients with HIT is discussed.

Abstract: Diagnosis and management of immune thrombocytopenic purpura (ITP) remain largely dependent on clinical expertise and observations more than on evidence derived from clinical trials of high scientific quality. One major obstacle to the implementation of such studies and in producing reliable meta-analyses of existing data is a lack of consensus on standardized critical definitions, outcome criteria, and terminology. Moreover, the demand for comparative clinical trials has dramatically increased since the introduction of new classes of therapeutic agents, such as thrombopoietin receptor agonists, and innovative treatment modalities, such as anti-CD 20 antibodies. To overcome the present heterogeneity, an International Working Group of recognized expert clinicians convened a 2-day structured meeting (the Vicenza Consensus Conference) to define standard terminology and definitions for primary ITP and its different phases and criteria for the grading of severity, and clinically meaningful outcomes and response. These consensus criteria and definitions could be used by investigational clinical trials or cohort studies. Adoption of these recommendations would serve to improve communication among investigators, to enhance comparability among clinical trials, to facilitate meta-analyses and development of therapeutic guidelines, and to provide a standardized framework for regulatory agencies.

Abstract: Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts due to accelerated platelet destruction and impaired platelet production. Thrombopoietin (TPO) is the major regulator of platelet production, and TPO mimetics have been approved to treat patients with ITP with a response rate around 80%. In this study, we assessed the impact of eltrombopag on patients at the RNA level and explored the biological mechanisms underlying the variations in individual responses through blood transcriptome profiling analysis. Methods: Peripheral blood specimens were collected for RNA extraction from patients with ITP at three time points: pre-treatment, one-week into treatment, and one-month into treatment. The criteria for response assessment are modified from the 2009 IWG guideline. The 3′-end RNA sequencing for expression quantification (3SEQ) was performed on 112 specimens from 37 patients. A globin-reduction approach was utilized for 77 samples with adequate RNA quantity to increase detection sensitivity. Sequencing data was mapped to human reference transcriptome to generate gene expression profiles. Using globin-reduced samples, SAMSeq analysis were conducted to identify differentially expressed genes. The pathways and upstream regulators associated with these genes were recognized using IPA (Ingenuity Pathway Analysis). With selected platelet specific genes as surrogate markers, post-treatment platelet gene expression- and platelet count-normalized platelet gene expression were compared with the pre-treatment expressions for each patient to assess platelet production and life span. In addition, the genomic coding regions of MPL (myeloproliferative leukemia virus oncogene) gene, encoding the receptor of TPO, were amplified by PCR and screened by Sanger sequencing for mutations in 10 responders and 7 nonresponders to assess for possible associations with drug responses. Results: No gene was identified as statistically significant to differentiate the responders and nonresponders at the pretreatment time point. No genetic variant within the coding regions of MPL was identified to be associated with the response to treatment. Along with the increased platelet counts at one-week into treatment, paired pretreatment and one-week into treatment analysis reveals TPO mimetics-induced upregulation of 215 genes in TPO responders (Figure 1A shows the SAM plot of SAMSeq two class paired comparison of 16 responders treated with eltrombopag at pretreatment and 1-week into treatment points, indicating a set of genes were upregulated at 1-week into treatment point. Figure 1C shows a heatmap of treatment-induced genes at baseline and after 1 week of eltrombopag treatment). On the contrary, nonresponders share no genes with statistically significant changes at one-week into treatment (Figure 1B). Interestingly, at one-month into treatment, responders show decreased expressions of the treatment-induced genes, even though the platelet counts are further elevated compared to the one-week into treatment time point. In responders, platelet gene expression was significantly increased at both one-week (p = 0.009) and one-month (p = 0.032) into treatment points, reflecting increased platelet production. Moreover, compared to the one-week into treatment time point, responders showed significantly reduced platelet count-normalized gene expression level (p = 0.033) at the one-month into treatment time point, indicating longer platelet life span and suggesting decreased platelet turnover rate. Pathway analysis identified TPO, GATA1 (GATA binding protein 1), and TGFB1 (transforming growth factor, beta1) as the top three activated upstream regulators for the treatment-induced genes. Conclusions: These results suggest that the TPO mimetics may exert dual effects in responders: stimulation of platelet production, as well as ameliorate platelet destruction, possibly involving TGFB1 pathway and regulatory T cells. Figure 1 SAMSeq analysis reveals treatment-induced gene expression in responders. Figure 1. SAMSeq analysis reveals treatment-induced gene expression in responders. Disclosures Bussel: Shionogi: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Zehnder:GlaxoSmithKline: Research Funding.

Abstract: Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

Abstract: Introduction: Cases of de novo immune thrombocytopenia (ITP), including a fatality following SARS-CoV-2 vaccination in a previously healthy recipient, led to studying its impact in pre-existing ITP. Published reports are limited but suggest that most patients with ITP tolerate the COVID-19 vaccines well without frequent ITP exacerbations (Kuter, BJH, 2021). Data regarding risk factors for exacerbation and relationship of response to first dose to that of second dose are limited. Methods: Data for patients with pre-existing ITP were obtained via 3 sources. First, via a ten-center retrospective study of adults with ITP who received a SARS-CoV-2 vaccine between December 2020 and March 2021 and had a post-vaccination platelet count (n=117); 9 centers were in the United States. Eighty-nine percent of patients received mRNA-based vaccines. The second and third sources of data were surveys distributed by the Platelet Disorder Support Association (PDSA) and the United Kingdom ITP Support Association. A 'stable platelet count' was defined as a post-vaccination platelet count within 20% of the pre-vaccination level. ITP exacerbation was defined as any one or more of: platelet decrease ≥ 50% compared to pre-vaccination baseline, platelet decrease by & gt;20% compared to pre-vaccination baseline with platelet nadir & lt; 30x10 9/L, receipt of rescue therapy for ITP. Continuous variables were described as mean ±SD or median [interquartile range]; categorical variables were described as n (%). Relative risks and 95% confidence interval were calculated to estimate strength of association. Results: Among 117 patients with pre-existing ITP from 10 centers who received a SARS-CoV-2 vaccine, mean age was 63±17 years, 62% were female, with median 12 [4-23] years since diagnosis of ITP; patients had received a median of 3 [2-4] prior medical treatments. Sixty-nine patients were on ITP treatment at the time of vaccination (Table 1). There was an almost even distribution of platelet count response following each vaccine dose. In 109 patients with data for dose 1, platelet counts increased in 32 (29%), were stable in 43 (39%), and decreased in 34 (31%); in 70 patients following dose 2, platelet counts increased in 24 (34%), were stable in 25 (36%), and decreased in 21 (30%) (Figure 1). Nineteen (17%) patients experienced an ITP exacerbation following the first dose and 14 (20%) of 70 after a second dose. In total, fifteen patients received and responded to rescue treatments (n = 6 after dose 1, n = 8 after dose 2, n = 1 after both doses). Of 7 patients who received rescue treatment after dose 1, 5 received dose 2 and only 1/5 received rescue treatment again. Rescue consisted of increased dose of ongoing medication, steroids, IVIG, and rituximab. Splenectomized persons and those who received 5 or more prior lines of medical therapy were at highest risk of ITP exacerbation. Only 1 of 47 patients who had neither undergone splenectomy nor received 5 or more lines of therapy developed ITP exacerbation after dose 1. There were 14 patients off-treatment at the time of dose 1 and 7 patients at time of dose 2; 1 patient in each group developed ITP exacerbation with both these having had normal counts prior to vaccination and having undergone splenectomy. In 43 patients whose platelet counts were stable or increased after dose 1 and received dose 2, only 6 (14%) had platelet decreases to & lt;50 x10 9/L after dose 2. Age, gender, vaccine type, and concurrent autoimmune disease did not impact post-vaccine platelet counts. In surveys of 57 PDSA and 43 U.K. ITP patients, similar rates of platelet change were seen (33% of participants reported decreased platelet count in both surveys) and prior splenectomy was significantly associated with worsened thrombocytopenia in each. Conclusions: Thrombocytopenia may worsen in pre-existing ITP post-SARS-CoV2-vaccination but when ITP exacerbation occurred, it responded well to rescue treatment. No serious bleeding events were noted. Rescue treatment was needed in 13% of patients. Proactive vaccination surveillance of patients with known ITP, especially those post-splenectomy and with more refractory disease, is indicated. These findings should encourage patients with ITP to not only be vaccinated, but to receive the second dose when applicable to ensure optimal immunization. Rituximab interferes with vaccination response and ideally would be held until a minimum of 2 weeks after completion of vaccination. Figure 1 Figure 1. Disclosures Lee: Principia Biopharma: Consultancy. Beltrami Moreira: Kadmon: Other: Spouse current employer; Jounce Therapeutics: Other: Spouse employment ended in the past 24 months. Al-Samkari: Amgen: Research Funding; Argenx: Consultancy; Agios: Consultancy, Research Funding; Dova: Consultancy, Research Funding; Rigel: Consultancy; Sobi: Consultancy; Novartis: Consultancy. Cuker: Novo Nordisk: Research Funding; Pfizer: Research Funding; Bayer: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; Novartis: Research Funding; UpToDate: Patents & Royalties; Alexion: Research Funding; Spark Therapeutics: Research Funding; Synergy: Consultancy. MacWhirter - DiRaimo: JD has not personally received any payment personally, but PDSA has received grants and consultancy fees from Novartis, grant and honorarium from Amgen, grant and consultancy fees from Pfizer and UCB, and grants from Argenx, Principia, Rigel, and CSL Behr: Consultancy, Honoraria, Other. Gernsheimer: Cellphire: Consultancy; Rigel: Research Funding; Principia: Research Funding; Novartis: Honoraria; Amgen: Honoraria; Dova: Consultancy; Sanofi: Consultancy. Kruse: Alex has not personally received any payment but reports that PDSA received grants and consultancy fees from Novartis, grant and honorarium from Amgen, grant and consultancy fees from Pfizer and UCB, and grants from Argenx, Principia, Rigel, and CSL Behri: Consultancy, Honoraria, Other. Kessler: Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Kruse: CK has not personally received payment but reports that PDSA received grants and consultancy fees from Novartis, grant and honorarium from Amgen, grant and consultancy fees from Pfizer and UCB, and grants from Argenx, Principia, Rigel, and CSL Behring: Consultancy, Honoraria, Other. Leavitt: Pfizer: Research Funding; Merck: Consultancy; CSL DOVA: Consultancy; Catalys: Consultancy; Behring: Consultancy; BPL: Consultancy; HEMA Biologics: Consultancy; Rigel: Consultancy; Syntimmune: Research Funding; Sangamo Therapeutics: Research Funding; BioMarin: Consultancy, Research Funding. Liebman: Sanofi/Genzyme: Research Funding; Novartis: Consultancy, Research Funding; Argenx: Research Funding; Amgen: Consultancy; Dova: Consultancy, Honoraria; Pfizer: Consultancy. Newland: Novartis: Consultancy, Research Funding, Speakers Bureau; UCB Biosciences: Consultancy; Roche: Speakers Bureau; Octapharma: Research Funding; GSK: Consultancy, Research Funding, Speakers Bureau; BMS: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Argenx: Consultancy, Speakers Bureau; Angle: Consultancy; Grifols: Consultancy, Speakers Bureau. Tarantino: Pfizer: Research Funding; Novo Nordisk: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; UCB: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Grifols: Research Funding, Speakers Bureau; Principia: Membership on an entity's Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dova: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMarin: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees. Thachil: Amgen: Speakers Bureau; Novartis: Speakers Bureau. Kuter: Up-to-Date: Patents & Royalties: Up-To-Date; Platelet Disorder Support Association: Membership on an entity's Board of Directors or advisory committees; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, BioCryst, Bristol Myers Squibb (BMS), Caremark, CRICO, Daiichi Sankyo, Dova, Genzyme, Immunovant, Incyte, Kyowa-Kirin, Merck Sharp Dohme, Momenta, Novartis, Pfizer, Principia, Protalex, Protalix, Rigel: Consultancy, Other: grant support and consulting fees; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, Bristol Myers Squibb (BMS), Immunovant, Kezar, Principia, Protalex, Rigel, Takeda (Bioverativ), UCB: Research Funding; Rubius: Current equity holder in publicly-traded company. Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board. Bussel: CSL: Other: DSMB; Dova/Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; UCB: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Momenta/Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; UptoDate: Honoraria; RallyBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia/Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.