We report here the first case of severe Immune thrombocytopenia induced by a secondgeneration cephalosporin antibiotic, Loracarbef. A 56-year old white female developed acute severe thrombocytopenia associated with acute respiratory symptoms following administration of Loracarbef. She responded to Loracarbef withdrawal and systemic corticosteroid administration. Loracarbef-dependent platelet-reactive antibodies were demonstrable in her serum by flow cytometry.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a low platelet count (less than 100 x 10^9 platelets/L) and an increased risk of bleeding. ITP is difficult to diagnose and manage due to the deficiencies in our understanding of the pathophysiological mechanisms leading to thrombocytopenia. Anti-platelet autoantibodies are believed to be the primary mechanism of thrombocytopenia in ITP. In this thesis, I demonstrate that autoantibodies can only be detected in half of all ITP patients; therefore, other mechanisms should be investigated. CD8+ T cells have been implicated as a mechanism of disease in ITP, but platelet-specific CD8+ T cells have yet to be identified. I have characterized CD8+ T cells in ITP patients and found that platelet-specific CD8+ T cells can be detected in ITP patients. These platelet-specific CD8+ T cells can also be detected in healthy individuals, so they are not specific to ITP. However, regulatory defects were observed in ITP patients and CD8+ T cell activity was elevated in ITP patients relative to healthy individuals and thrombocytopenic non-ITP patients. Investigating whether platelet-specific CD8+ T cells can actively participate in platelet destruction and underproduction will be an essential step towards better understanding the role of CD8+ T cells as a disease mechanism in ITP, which will lead to improvements in the management of ITP. / Thesis / Doctor of Philosophy (PhD) / Platelets are small blood cells that are involved in minimizing blood loss at the site of a wound by forming a plug. In a disease called immune thrombocytopenia (ITP), patients have a low platelet count, which can result in bleeding. The bleeding symptoms of ITP decrease the quality of life for ITP patients and can be life-threatening in rare cases. It is believed that ITP is caused by proteins produced by the immune system called antibodies. I found that the antibodies that cause ITP can only be detected in half of all ITP patients. Therefore, there are probably additional causes of ITP. It is suspected that CD8+ T cells might cause ITP in some patients. CD8+ T cells are part of the immune system and they typically destroy other cells that are cancerous or infected by viruses. CD8+ T cells might also destroy healthy cells, like platelets. My goal was to characterize CD8+ T cells in order to determine their role in ITP. I found that CD8+ T cells from ITP patients can target platelets, and that healthy people have these CD8+ T cells as well. In regard to CD8+ T cells that target platelets, the difference between ITP patients and healthy people appears to be related to immune system regulation and CD8+ T cell activity. In the future, we should focus on understanding how platelet-specific CD8+ T cells can cause a low platelet count in order to improve the clinical management of ITP.

The day that I joined McMaster University and the Department of Health Research Methods, Evidence, and Impact as a Master student, I did not foresee the amazing journey I was embarking on or the impact of my academic achievements. I was continuously challenged to think critically and apply my ideas to real world problems. Seeing the opportunity to make a difference in clinical research and patient care inspired me to begin my PhD studies. Creating knowledge and applying it practically was a difficult task, however, the opportunities to share my research in a dynamic and complex field with the world fuelled my motivation. Completing a PhD has been an incredible privilege for which I will always be grateful. To my family – Mamma, Papa, Mark and Matthew – thank you for your undying and unconditional love, support, and encouragement. A pandemic forced us to come together, and having your support 24/7 (literally) made the journey more enjoyable. To my closest friends, thank you for being a part of my life and supporting my ambitions. We all recognized my ambitiousness would set me on a difficult path and completing a PhD was not my original plan. Thank you for inspiring me to take on the challenge and ensure my work was truly impactful. Thank you for encouraging mental strength, being understanding and willing to lend a hand, reminding me that I can achieve anything I set my mind to, and inspiring me to dare. To my supervisory committee members and independent study supervisor, thank you for your continued interest in my work and for always supporting my ambitions. I am grateful for your patience and belief that I would succeed in completing what I had sought to achieve. Thank you for the chance to work together, and for your constant support and mentorship throughout my PhD journey. To the faculty, staff and fellow students at the Department of Health Research Methods, Evidence, and Impact, and the McMaster Centre for Transfusion Research, thank you for providing the resources, friendship, and guidance I needed to achieve greatness. To the panel members of the ITP Emergency Management Guideline, thank you for believing in this project and making this work possible. I look forward to implementing the results of our efforts into clinical practice. To the ITP patient community, thank you for entrusting me with the task of improving patient care and supporting me along the way. You have enabled me to be a voice for change. To the rare disease community, I know the completion of my PhD journey will not be the end of our work together. To the funding agencies who allowed this project to be possible, the Canadian Institutes of Health Research and Platelet Disorder Support Association, thank you for supporting my PhD journey and the completion of this important work. To my doctoral supervisor, Dr. Donald Arnold, it has been an honour and privilege to have learned from you and received your guidance throughout my PhD journey. Since our initial meeting, when I hobbled into the McMaster Centre for Transfusion Research offices several minutes late, I felt that your kindness and honesty would be the perfect form of mentorship to push me towards success. Thank you for recognizing my ambition and reminding me to keep focused. I will continue to apply this lesson throughout my life and strive for ‘depth’. For many years, you have been my mentor who I reached out to for advice, support, honest feedback, and encouragement. Thank you for imparting your knowledge to me over my PhD journey and teaching me how to be an inspirational mentor who highlights the strengths of their students while simultaneously supporting their growth. Although I may have finally reached the destination in my PhD journey, I know that we will continue to collaborate for many years to come. / Developing clinical practice guidelines (CPGs) for rare diseases is methodologically challenging. As each disease has so few patients, published literature includes low-quality studies or studies that do not directly address the questions of interest. As a result, CPG panelists have limited evidence on which to base their recommendations. Historically, when no evidence was available, CPGs have relied on physician opinion. This does not align with the mandate of CPGs which transparently identifies, appraises, and relies on evidence. The challenges of developing CPGs for rare diseases are exemplified by immune thrombocytopenia (ITP), a rare autoimmune disease that affects approximately 1 in 8,000 people. It predominantly affects females and young adults, and is characterized by low blood platelets that increase the risk of bleeding. Bleeding emergencies in ITP patients are critical, life-threatening events that can cause life-long morbidity and associated health care costs. Treatment of ITP bleeding emergencies requires a rapid, coordinated approach that involves emergency department staff, hematologists, pharmacy, and the laboratory. However, there is no evidence-based CPG for the management of ITP bleeding emergencies. The objectives of my PhD thesis are (1) exploring the heterogeneity of ITP diagnosis using antiplatelet autoantibodies; (2) developing a standardized definition of ITP bleeding emergencies; (3) outlining the synthesis of existing evidence on the treatment of ITP bleeding emergencies through a systematic review; and (4) developing a novel methodology to address the lack of evidence in rare disease CPGs and applying it to develop a CPG for the management of ITP bleeding emergencies. / Thesis / Candidate in Philosophy / Guidelines for rare diseases can be hard to develop because of a lack of information. Doctors and researchers make decisions on rare disease management based on their experiences, which can be limited. Low blood platelets and emergency bleeding can be caused by a rare disease called immune thrombocytopenia (ITP). When emergency bleeds occur, patients need care from the Emergency Department immediately. The problem is that there is no standard way for doctors to treat these ITP bleeding emergencies. My PhD thesis project will fill an important gap for ITP emergency treatment. First, we will assess how ITP patients are diagnosed. Second, we will define an ITP bleeding emergency. Third, we will collect existing information about ITP bleeds. Fourth, we will overcome the challenge of not having enough information by collecting new data from patient records. The method we use to develop ITP guidelines can be used for other rare diseases.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Immune thrombocytopenia (ITP) is an autoimmune disorder, mediated mainly by autoantibodies against platelet glycoprotein GPIIbIIIa and GPIbα resulting in enhanced platelet destruction. Decreased platelet production and cellular immunity also contribute to ITP. GPIIbIIIa and GPIbα are distinct platelet receptors. Previous studies suggested that anti-GPIbα (versus anti-GPIIbIIIa)-mediated ITP is less responsive to IVIG therapy. However, little information is available whether antibody specificities also dictate efficacy of Glucocorticosteroids (GC), which are the first-line ITP treatment. Here, I first induced ITP in mice by passive administration of anti-GPIbα or anti-GPIIbIIIa antibodies. Results suggest GCs were more effective at amelioration of anti-GPIIbIIIa-mediated thrombocytopenia. I repeated this observation in an active ITP model, in which splenocytes from wild-type platelet immunized GPIbα-/- or GPIIIa-/- mice were engrafted into wild-type mice, which developed ITP. Thus, I established new murine models of ITP for GC therapy and demonstrated that anti-GPIbα-mediated thrombocytopenia may be less responsive to GC therapy.

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder which results from fetal platelet destruction by maternal antibodies against platelet antigens, including GPIIbIIIa (αIIbβ3 integrin) and GPIbα. β3 integrin is also expressed by angiogenic endothelial cells (ECs) and is required for angiogenesis. Therefore, we investigated whether anti-β3 antibodies in FNIT cross-react with blood vessels of the fetus/neonate and contribute to pathogenesis. Antibodies to GPIbα were used as controls. To mimic human FNIT, β3 integrin- or GPIbα-deficient female mice were immunized with wild-type platelets and bred with wild-type male mice. Pups in both groups had thrombocytopenia but intracranial hemorrhage was only observed in anti-β3-mediated FNIT. Anti-β3-mediated FNIT pups had increased apoptosis in the brain and impaired vascularization of the brain and retina. In addition, anti-β3 sera inhibited proliferation and vascular-like tube formation by ECs in vitro. Therefore, anti-β3 antibodies in FNIT likely impair angiogenesis in the developing fetus/neonate.

Immune thrombocytopenia (ITP) is an autoimmune disorder, mediated mainly by autoantibodies against platelet glycoprotein GPIIbIIIa and GPIbα resulting in enhanced platelet destruction. Decreased platelet production and cellular immunity also contribute to ITP. GPIIbIIIa and GPIbα are distinct platelet receptors. Previous studies suggested that anti-GPIbα (versus anti-GPIIbIIIa)-mediated ITP is less responsive to IVIG therapy. However, little information is available whether antibody specificities also dictate efficacy of Glucocorticosteroids (GC), which are the first-line ITP treatment. Here, I first induced ITP in mice by passive administration of anti-GPIbα or anti-GPIIbIIIa antibodies. Results suggest GCs were more effective at amelioration of anti-GPIIbIIIa-mediated thrombocytopenia. I repeated this observation in an active ITP model, in which splenocytes from wild-type platelet immunized GPIbα-/- or GPIIIa-/- mice were engrafted into wild-type mice, which developed ITP. Thus, I established new murine models of ITP for GC therapy and demonstrated that anti-GPIbα-mediated thrombocytopenia may be less responsive to GC therapy.

Immune thrombocytopenia (ITP) is an autoimmune hematologic disorder characterized by a low platelet count due to increased platelet destruction or decreased production. In primary ITP, the patient can have a low platelet count (<100 billion cells/L) for clinically unknown reasons. ITP is a rare disease that affects approximately 3/100 000 adults each year and some patients may experience bleeding symptoms. Autoantibody-mediated autoimmunity plays a role in the destruction of platelets by targeting platelet glycoproteins (GPs). Autoantibodies against platelet membrane GPIIbIIIa and GPIbIX are observed in about 50% of patients through direct antigen-capture assays, and 18% in patients through indirect antigen-capture assays. It is possible that some antibodies may not be detectable due to affinity or titre, or there may be other factors involved in platelet destruction. Currently, there is no definitive diagnostic test available for ITP, as a result of low assay sensitivity and different mechanisms involved in disease pathogenesis. The objective of this study was to use a novel approach to increase autoantibody detection unique to ITP patients. Total IgG was purified from patient and control plasma samples. A streptavidin-based antigen-capture assay was optimized to test the effect of biotinylation on the detection of anti-GPIIbIIIa and anti-GPIbIX autoantibodies in primary ITP patients (n=14), secondary ITP patients (n=3), non-immune thrombocytopenic controls (n=2) and healthy controls (n=16). Streptavidin-coated biosensors were used in an optimized biolayer interferometry (BLI) assay to study autoantibodies binding to biotinylated GPIIbIIIa and GPIbIX. Detection of anti-GPIIbIIIa autoantibodies in the streptavidin antigen-capture assay had a sensitivity of 24% and anti-GPIbIX autoantibodies had a sensitivity of 25%. BLI showed binding of autoantibodies in approximately 5% of ITP samples for both GPIIbIIIa and GPIbIX. The samples that had detectable autoantibodies in the antigen-capture assay did not have detectable antibodies in the BLI assay. BLI was not able to confirm antibody detection found in enzyme immunoassays. / Thesis / Master of Science (MSc) / Platelets are blood cells involved in clotting at sites of injury. Immune thrombocytopenia (ITP) is a disease defined by a low platelet count that can lead to bleeding. ITP is a rare disease that affects 3 in 100 000 adults every year. ITP is thought to be caused by proteins known as antibodies that bind self-platelets and lead to their destruction. These antibodies are directly found on approximately 50% of patients’ platelets, and only 18% of patients have antibodies in circulation. It is possible in many patients, antibodies are present at a low concentration, or are too weak to be detected in antibody tests. In this study, a new technology known as biolayer interferometry was employed to find antibodies in a higher percentage of patients. Results showed only 6% of ITP patients had detectable antibodies in their circulation. This research will improve our understanding of antibodies in ITP.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder, characterized by platelet destruction and/or underproduction. The pathophysiology is heterogeneous and can be mediated by autoantibodies and cytotoxic T lymphocytes (CTLs). While platelet destruction in ITP is well documented, there is little support for platelet underproduction due to the inhibition of megakaryocyte growth and considerably less support for CTL-mediated platelet underproduction. Our objective was to develop an assay that could test for CTL-mediated inhibition of megakaryocyte growth (megakaryopoiesis) in ITP, using healthy controls. Peripheral blood from healthy donors was used to prepare hematopoietic stem and progenitor cells (HSPCs). These cells were expanded with StemSpan to culture a large number of megakaryocytes for the CTL assay. Our studies show that CTLs can be stimulated in-vitro using anti-CD3 antibodies and that they can be used after freezing and thawing. We also assessed CTL stimulation via peptide presentation, using viral peptides whom almost 100% of the general population have memory CTL specificity to, in order to activate a lower frequency of CTLs and to model levels of CTL activation in autoimmune disease. Both stimulants were found to stimulate CTLs in healthy donors with donor variability in the IFN-γ ELISpot. The CTL assay was developed by co-culturing thrombopoietin (TPO) stimulated HSPCs with autologous CTLs for 7 days to observe inhibition of megakaryocyte growth. To induce CTL stimulation, CTLs were either incubated with anti-CD3 or HSPCs were incubated with viral peptides before co-culturing with CTLs. Results showed that while viral peptides can be used as an internal control for the CTL assay, it could not serve as a positive control as inhibition was donor dependent. Inhibition of megakaryocyte growth in the presence of anti-CD3 stimulated CTLs was observed in all donors, validating its use as an appropriate positive control to study CD8+ T cell pathophysiology in ITP. / Thesis / Master of Science (MSc)

Defined as a platelet count <100x109/L with no known cause, immune thrombocytopenia (ITP) is a diagnosis of exclusion, meaning other thrombocytopenic conditions must be ruled out before establishing the ITP diagnosis. This can lead to errors, unnecessary exposures to expensive and harmful treatments, and increased patient anxiety and distress. In the absence of a standardized diagnostic test, a clinical prediction model, called the Predict-ITP tool, was developed to aid hematologists in establishing the ITP diagnosis among patients who present with thrombocytopenia. Based on a cohort of 839 patients referred to an academic hematology clinic and using penalized logistic regression, the following predictor variables for the ITP diagnosis were identified: 1) high platelet variability index; 2) lowest platelet count; 3) highest mean platelet volume; and 4) history of a major bleed. Internal validation was completed using bootstrap resampling, and showed good discrimination and excellent calibration. Following internal validation and prior to implementation, the Predict-ITP Tool must undergo external validation by evaluating the tool’s performance in a different cohort. A study protocol was developed with the objective of externally validating the Predict-ITP Tool by collecting data from 960 patients from 11 clinics across Canada. The tool will compute the probability of ITP using information available at the time of the initial consultation, and results will be compared with either the local hematologist’s diagnosis at the end of follow-up or the adjudicated diagnosis. Discrimination (the ability to differentiate between patients with and without ITP) and calibration (the agreement between predicted and actual classifications) of the tool will be assessed. The Predict-ITP Tool must demonstrate good discrimination (c-statistic ≥ 0.8) and excellent calibration (calibration-in-the-large close to 0; calibration slope close to 1) to achieve external validation. If implemented, this tool will improve diagnostic accuracy and reduce delays in diagnosis and unnecessary treatments and investigations. / Thesis / Master of Science (MSc) / There lack of a standardized test to diagnose immune thrombocytopenia (ITP) leads to delays in care, use of incorrect treatments, and increased patient anxiety. The Predict-ITP Tool was developed to classify patients as ITP or non-ITP using the following data: 1) platelet counts in the recent past; 2) the highest mean platelet volume; and 3) major bleeding at any time in the past. The preliminary internal validation study showed promise. I developed a study protocol to externally validate the Predict-ITP Tool that will collect data from 960 patients from 11 clinics across Canada to see how accurately the tool would have performed to classify patients as ITP or non-ITP at the first hematology visit compared with the gold standard clinical diagnosis by the hematologist or an independent expert committee. A successful external validation that demonstrates the tool’s predictive accuracy in an external population must be completed before widespread use.