The course of the haemopoietic response during canine babesiosis caused by Babesia rossi has not previously been studied. This prospective, descriptive longitudinal study on clinical cases describes the haematological kinetics during the first six days following treatment of natural babesiosis infection. Ninety client-owned dogs diagnosed with B rossi infection, based on examination of a Cam’s Quick-Stain-stained thin capillary blood smear and confirmed by polymerase chain reaction analysis, were included. At first consultation, 24 hours, three days and six days after first consultation, or until death, an EDTA sample was collected from the jugular or cephalic vein and submitted for a full automated blood count, using a CELL-DYN 3700 analyzer. Manual leukocyte differential counts were performed. Based on the treatment protocol, the dogs were divided into a blood transfusion group, and a non blood transfusion group. A slightly to moderately regenerative normocytic normochromic anaemia occurred throughout the study period for both treatment groups. The anaemia was very severe at presentation in dogs that received a blood transfusion and moderate at presentation in dogs that did not receive a blood transfusion. Anaemia was still present by the end of the study period in both treatment groups. The regenerative response was moderate in severely anaemic dogs and mild in moderately anaemic dogs. A mild inflammatory leukocytic response was found in both treatment groups. The median segmented neutrophil count for both treatment groups was within the reference interval throughout the study period. A left shift occurred more commonly in dogs that received a blood transfusion, and was significantly influenced by the degree of anaemia at presentation. In dogs with a left shift, a degenerative left shift, not influenced by the degree of anaemia at presentation, was found more commonly. Severe thrombocytopaenia for both treatment groups, which resolved within a week in both groups, was found. Treatment with a blood transfusion reduced the anaemia, but had no significant effect on white blood cell or platelet responses. Blood cell responses were not significantly influenced by age, previous infection with babesiosis or duration of illness. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2008. / Companion Animal Clinical Studies / unrestricted

Valproic acid (Depakote) is an antiepileptic drug approved for the treatment of bipolar disorder, migraine prophylaxis, and seizure disorders. While the exact mechanism is still unknown, thrombocytopenia, defined as platelet counts < 150,000/uL, has been reported secondary to Depakote treatment. The frequency of Depakote-induced thrombocytopenia varies greatly, with reported rates ranging from 5 to 54%. This adverse effect is dose-dependent and possible risk factors include lower baseline platelet counts, female gender, and high VPA serum concentrations.Our team came across two patient cases where thrombocytopenia during Depakote therapy was observed. Patient information was gathered through electronic medical records. The first patient was a 65-year-old male who was started on 500 mg Depakote ER three tablets at night for bipolar affective disorder. After several months on this dose, the patient’s platelets decreased to 59 X 103per microliter. One month after the drug was discontinued, the platelets recovered to 160 X 103per microliter. The second patient was a 57-year-old woman who had two occurrences of thrombocytopenia while on Depakote. The patient was started on Depakote for a seizure disorder. She was later admitted for symptomatic bradycardia, hypotension, and concern for thrombocytopenia. Her Depakote dose was decreased from 500 mg three times a day to twice a day. Approximately 5 weeks later, she presented to the emergency room for decreased arousal and hypotension. She was again found to have thrombocytopenia with a platelet count of 28 X 103per microliter with a Depakote level of 101 mcg/mL. The team discovered she had been receiving Depakote 500 mg three times a day following discharge from her last admission, not the reduced dose prescribed. On day four of admission, her platelets had not improved and the Depakote dose was decreased further to 250 mg twice daily. After Depakote was discontinued her platelets gradually improved and returned to normal after four days, the eighth day of admission. Utilizing the Naranjo adverse drug reaction probability scale, the first patient case had a probable reliability that this adverse reaction was due to Depakote, while the second patient case had a definite reliability.These cases illustrate the potential for thrombocytopenia secondary to Valproic acid use. Although this adverse event isn’t well understood, these cases add to the evidence that it can occur. Recognition of this reaction is important and clinicians should monitor hematologic labs, including platelets, for patients receiving Valproic acid.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21693号 / 医科博第97号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 中川 一路, 教授 朝長 啓造, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

No / The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1(CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.

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)

Background: Heparin-induced thrombocytopenia (HIT) is an iatrogenic immune-mediated prothrombotic disorder that is a direct consequence of heparin therapy. In HIT, antibodies are generated against complexes of platelet factor-4 (PF4) and heparin. Immunoglobulin G (IgG) antibodies bind to PF4/heparin complexes and cause Fc-receptor-mediated activation of platelets and monocytes. PF4 binds endogenous heparin-like polyanions to reveal cross-reactive epitopes that can also bind anti-PF4/heparin antibodies. Based on this observation, researchers have suggested that exposure to PF4/polyanion complexes can sensitize immune cells to become activated to produce HIT antibodies following iatrogenic heparin exposure. Research objective: The objective of this study is to determine whether the preoperative PF4-dependent immune response is associated with postoperative anti-PF4/heparin antibody production in a cohort of patients undergoing cardiopulmonary bypass surgery. Materials and methods: To assess the preoperative immune response to PF4, we utilized two assays: (1) a 3H-thymidine uptake assay to measure peripheral blood mononuclear cell (PBMC) proliferation in response to in vitro stimulation with PF4 and (2) a PBMC ELISPOT assay to measure the preoperative frequency of PF4-specific antibody-secreting cells. Proliferation was quantified as a stimulation index (SI). We then utilized a PF4/heparin-dependent enzyme immunoassay to measure the in vivo levels of anti-PF4/heparin antibodies produced by these patients in the postoperative period. Results: Our findings suggest that preoperative PF4-dependent proliferation is not associated with postoperative polyspecific anti-PF4/heparin antibody production [Spearman’s ρ (95% CI) = –0.02 (–0.32, 0.28), P = 0.91]. PF4-dependent proliferation had a weak negative association with postoperative anti-PF4/heparin IgG antibody production [Spearman’s ρ (95% CI) = –0.31 (–0.56, –0.02), P = 0.04], but was not associated with postoperative IgM or IgA anti-PF4/heparin antibody production [IgM: Spearman’s ρ (95% CI) = –0.04 (–0.33, 0.26), P = 0.78; IgA: Spearman’s ρ (95% CI) = –0.05 (–0.34, 0.25), P = 0.73]. Qualitative analysis demonstrated that two patients who had the strongest preoperative PF4-dependent proliferation responses produced the highest postoperative levels of anti-PF4/heparin IgM antibodies, but this relationship was not observed with postoperative anti-PF4/heparin IgG antibodies. Moreover, the preoperative frequency of PF4-specific antibody-secreting cells (ASCs) was also not associated with postoperative levels of anti-PF4/heparin IgM or IgG antibodies [IgM: Spearman’s ρ (95% CI) = 0.30 (–0.79, 0.93), P = 0.683; IgG: Spearman’s ρ (95% CI) = –0.21 (–0.92, 0.83), P = 0.600]; however, this was only completed on five patients and so the sample size should be increased before any meaningful conclusions can be drawn. We also demonstrated that PF4-dependent proliferation increases 5–6 days following cardiopulmonary bypass surgery [geometric mean (GM) postoperative PF4 alone proliferation (in SI) vs. GM preoperative PF4 alone proliferation (in SI) ± SEM: 23.7 ± 1.3 vs. 6.9 ± 1.5, P = 0.009]. Conclusions: Based on our findings, we conclude that preoperative PF4-dependent proliferation is unable to predict postoperative anti-PF4/heparin antibody production in this cohort of cardiopulmonary bypass patients. Due to the small sample size, we are unable to make conclusive statements regarding the relationship between preoperative PF4-specific ASC frequency and postoperative anti-PF4/heparin antibody production, but our findings would suggest that an association does not exist between these two variables in this patient cohort. Cardiopulmonary bypass surgery, however, may mobilize the postoperative immune cell repertoire to become activated against the self-protein PF4 and may therefore contribute to the postoperative HIT immune response. / Thesis / Master of Science (MSc) / Background: Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that is a direct consequence of heparin therapy. In HIT, antibodies are generated against complexes of platelet factor-4 (PF4) and heparin. Antibodies bind to PF4/heparin complexes and cause activation of platelets and monocytes. Researchers have suggested that exposure to PF4/polyanion complexes can sensitize immune cells to become activated to produce HIT antibodies following iatrogenic heparin exposure. Research objective: The objective of this study is to determine whether the preoperative PF4-dependent immune response is associated with postoperative anti-PF4/heparin antibody production in a cohort of patients undergoing cardiopulmonary bypass surgery. Materials and methods: To assess the preoperative immune response to PF4, we measured cellular proliferation in response to PF4 stimulation and the preoperative frequency of PF4-specific antibody-secreting cells. We also measured the level of anti-PF4/heparin antibodies following surgery. Results: Our findings suggest that preoperative PF4-dependent proliferation is not associated with postoperative anti-PF4/heparin antibody production. Moreover, the preoperative frequency of PF4-specific antibody-secreting cells (ASCs) was also not associated with postoperative levels of anti-PF4/heparin antibodies; however, this was only completed on five patients and so the sample size should be increased before any meaningful conclusions can be drawn. We also demonstrated that proliferation increases 5–6 days following cardiopulmonary bypass surgery. Conclusions: Based on our findings, we conclude that preoperative proliferation is unable to predict postoperative anti-PF4/heparin antibody production in this cohort of patients. Due to the small sample size, we are unable to make conclusive statements regarding the relationship between preoperative ASC frequency and postoperative anti-PF4/heparin antibody production. Cardiopulmonary bypass surgery, however, may mobilize the postoperative immune cell repertoire to become activated against the self-protein PF4 and may therefore contribute to the HIT immune response.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

Haematological malignancies and their treatment lead to prolonged periods of severe thrombocytopenia (platelet count ≤ 50 x 10⁹/l). Despite the use of prophylactic platelet transfusions, haemorrhage remains an important complication during this thrombocytopenic period. Within a 30 day period up to 70% of patients have clinically significant haemorrhage (World Health Organization (WHO) grade 2 or above bleeding) and up to 10% have severe or life-threatening haemorrhage (WHO grade 3 or 4 bleeding). Hence our current management of these patients to prevent haemorrhage is sub-optimal. The aim of this thesis was to identify clinical and laboratory factors that may predict the risk of haemorrhage in patients with haematological malignancies and severe thrombocytopenia. This was achieved via several different study designs and assessed the effect of clinical and laboratory factors on any or clinically significant haemorrhage and their effect on intracranial haemorrhage. This thesis has demonstrated that there is no consensus on how bleeding is assessed and graded in this patient group. Also it showed that the absolute immature platelet number may be a better alternative to the total platelet count to guide administration of platelet transfusions. Female sex, a previous history of a fungal infection, a high C-reactive protein, a high white cell count, a low platelet count, anaemia, impaired renal function, and recent clinically significant haemorrhage were all found to be independent risk factors for haemorrhage. Patients who were in complete remission from their haematological malignancy had a much lower risk of bleeding.

Résumé : Aux soins intensifs (SI), une diminution du décompte plaquettaire peut avoir un impact important. Des études ont démontré une association entre la thrombopénie et la durée de séjour, les saignements, les transfusions et la mortalité. Les facteurs qui prédisent une thrombopénie varient selon les populations étudiées. Les médicaments sont souvent suspectés, mais peu ont été indépendamment associés à la thrombopénie aux SI. Les études publiées à ce jour souffrent de plusieurs limites. Les objectifs de cette thèse étaient de décrire l’épidémiologie, identifier les facteurs de risque associés, d’évaluer l’impact sur la morbidité et la mortalité et d’évaluer les causes indépendantes médicamenteuses de thrombopénie aux SI. Une cohorte rétrospective a été créée à partir de données administratives et cliniques afin de décrire l’épidémiologie, et d’évaluer les facteurs prédictifs et les conséquences de la thrombopénie aux SI. Par la suite, une étude cas-témoin a été entreprise en sélectionnant les patients qui ont souffert d’une thrombopénie de cause indéterminée afin de déterminer les associations entre les médicaments fréquemment utilisés aux SI et la thrombopénie. Un total de 20 711 patients a été inclus dans l’analyse. La prévalence et l’incidence de thrombopénie ont été de 13,3% et 7,8%, respectivement. La thrombopénie a été indépendamment associée à une augmentation des saignements majeurs (aRC 1,32 95% CI 1,20-1,46). Après des ajustements statistiques, la thrombopénie était associée à une augmentation de mortalité (aRC 1,25 IC95% 1,20-1,31). L’impact sur la mortalité a été le plus important dans les catégories d’admission suivantes: cancer, respiratoire, digestif, génito-urinaire et infectieuses. Les facteurs de risque indépendants suivants ont été identifiés: l’âge, le genre masculin, la ventilation mécanique, l’alcoolisme, la cirrhose hépatique, le décompte plaquettaire à l’admission, l’hypersplénisme, le ballon intra-aortique, le choc septique, l’hépatite aiguë, la chirurgie de pontage coronarien et les maladies thromboemboliques. Dans l’étude cas-témoin, 200 cas de thrombopénie ont été identifiés après l’exclusion des maladies fortement associées. Ces cas ont été appariés à 200 témoins admis dans la même année. Parmi les 15 classes de médicaments évalués, seules les quinolones 1,67 (IC95% 1,00-2,87) ont été indépendamment associées à la thrombopénie dans le modèle final. En conclusion, la thrombopénie est indépendamment associée à une hausse de la mortalité qui varie grandement selon la catégorie d’admission. Les facteurs de risque sont nombreux et incluent des facteurs modifiables. Bien que les médicaments soient fréquemment soupçonnés, seules les quinolones semblent être associées à la thrombopénie aux SI. // Abstract : In the intensive care unit, a reduction in platelet counts can have a major impact on patient outcomes. Studies have showed an association between thrombocytopenia and length of stay, bleeding, blood product administration and mortality. Predictors of thrombocytopenia in the intensive care setting vary according to studies. Although medications are often suspected as potential causes, few have been independently associated with thrombocytopenia. In addition, published studies have many limits including small sample sizes, probable residual confounding, and inclusion of invasive interventions. The objectives of the thesis are to describe the epidemiology of thrombocytopenia, identify its risk factors, evaluate its impact on morbidity and mortality, and evaluate the drug-induced causes of thrombocytopenia. A retrospective cohort was created using administrative and clinical data. In the first study, multivariate analysis was used to identify risk factors. In the second study, a case control strategy was used to determine the association between thrombocytopenia and drugs commonly used in the intensive care unit previously associated with thrombocytopenia. A total of 20 711 patients were included in the analysis. The prevalence and incidence of thrombocytopenia defined as a platelet count below 100 x 10[superscript 9]/L were 13.3% and 7.8%, respectively. Thrombocytopenia was independently associated with an increase in the risk of major bleeding (aOR 1.32 95% CI 1.20-1.46). After adjusting for confounders, thrombocytopenia was associated with an increased risk of hospital mortality (aOR 1.25 IC95% 1.20-1.31). The impact of thrombocytopenia on mortality was the most important in the following diagnostic categories: cancer, respiratory, digestive, genitourinary, and infectious. The following independent risk factors were identified: age, male gender, admission platelet counts, mechanical ventilation, alcoholism, liver cirrhosis, hypersplenism, intra aortic balloon pumps, septic shock, acute hepatitis, coronary bypass surgery and venous thromboembolism. In the case control study, 200 cases were identified after exclusion of patients with diseases strongly associated with thrombocytopenia and paired with 200 controls admitted in the same year. Among the 15 medication classes evaluated, only quinolones 1.67 (IC95% 1.00-2.87) were independently associated with thrombocytopenia. In conclusion, thrombocytopenia is independently associated with an increased risk of mortality, which varies according to diagnostic admission categories. Risk factors are numerous and some are modifiable. Although medications are often suspected, only quinolones were statistically associated with thrombocytopenia.