Stem cell transplantation is a known treatment for various cancers. Currently most cells transplanted are collected via apheresis. An injection of growth factor is given to the patient to start the proliferation and mobilization of stem cells. Apheresis can be initiated when the patient has a stem cell count of 15 to 20 stem cells/µL of peripheral blood. The standard method with which stem cells are analysed is immune flow cytometry where CD34+ and CD45+ are identified with targeted fluorescent antibodies. This analysis takes more than 45 minutes to perform.     Sysmex XN-9000 analyses samples with flow cytometry by lysing erythrocytes and platelets and staining the leukocytes with fluorescent dye. Analysis of the hematopoietic progenitor cells (HPC) takes less than 4 minutes. The purpose of this study was to investigate ifit is possible to predict the start of the apheresis using XN-9000.     For this study, 43 samples were analysed using both methods. Using the sign test, a p-value was calculated to <0.05, which indicates a significant difference between the results received by the two methods. Spearman’s rank correlation gave an observed ρ-value > the critical ρ-value which revealed a correlation between the methods, although not linear according to Pearson’s correlation coefficient. PPV and NPV were calculated with cut-off at 20, 30 and 40 HPC/µL blood where 20 HPC/µL gave an NPV at 100 %. According to the test made, there is correlation between the two methods, but further samples must be analysed to investigate how the results should be compared.

Background: Bernard Soulier Syndrome (BSS) is a rare, autosomal recessive inheritance disorder of platelet function. Estimated to affect one per one million, there are currently only 200 cases reported worldwide presenting more commonly in families with parental consanguinity. This syndrome occurs when there is a genetic defect in the subunits (GPIb-alpha, GPIB-beta, and GP9) that form the GPIb-IX-V complex. The result is inadequate binding to von Willebrand factor. The clotting cascade is, therefore, unable to begin, causing symptoms of excessive and prolonged bleeding. Objectives: We report a case with multiple episodes of exaggerated bleeding and easy bruising. Methods: We analyzed complete blood count, coagulation studies, platelet aggregation assays, platelet glycoprotein expression by flow cytometry, as well as screened both patient and parents for relevant genes responsible for BSS. Results: 14-month-old Caucasian male born at 38w3d gestational age, non-consanguineous parents with multiple episodes of exaggerated bleeding and easy bruising from minor injuries. His symptoms started early in life with excessive bleeding after circumcision. No history of intramuscular, joint, or intracranial bleeding. Complete blood counts showed macrothrombocytopenia (98 X109 /L MPV 12.3 fl) no leukocyte inclusion bodies on peripheral smear. Coagulation tests (prothrombin time, activated partial thromboplastin time, vWF antigen, and vW-Ristocetin cofactor activity, platelet function assay) were normal. Platelet glycoprotein expression by flow cytometry revealed significantly reduced binding of monoclonal antibodies to platelet GPIb and normal GPIIb/IIIa. Comprehensive platelet disorder panel revealed two clinically significant variants missense mutations in the GP9 gene (P.Cys135 Tyr and P.Asn61Ser) These variants were on opposite alleles and results were consistent with the diagnosis of Bernard Soulier syndrome (BSS). The mother reported heavy menstrual cycles, the father had no significant bleeding symptoms, and both parents had normal platelet counts. Target genetic testing identified these two distinct missense mutations from both Mother and Father of the child. Conclusion: The two rare variants occurring on the gene for GPIX (GP9) increase the number of known genetic defects associated with the manifestation of Bernard Soulier Syndrome.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13109号 / 論医博第2127号 / 新制||医||1022(附属図書館) / (主査)教授 今中 雄一, 教授 川上 浩司, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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.

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<p>Venous thromboembolism (VTE), which encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE), is the most preventable cause of death in hospitalized patients. Due to its high mortality, morbidity, and cost, health care providers are obligated to not only effectively diagnose and treat VTE, but also to prevent it if possible. This has been reinforced by a number of national and international quality initiatives to prevent hospital-acquired VTE. Despite the existence of well-accepted clinical practice guidelines on VTE prophylaxis, 1 in 3 hospitalized medical patients receives an inappropriate VTE prophylaxis strategy. Both underuse of prophylaxis in patients with VTE risk, and overuse of prophylaxis in patients without VTE risk are problems. The use of inappropriate VTE prophylaxis strategies is likely due to the complexity and heterogeneity of hospitalized medical patients, and the difficulty of applying “one size fits all” practice guidelines to this group. Institution-wide knowledge translation strategies are required to close the gap between evidence and practice, and promote evidence-based VTE prophylaxis strategies in hospitalized medical patients. The objective of this thesis is to design a cluster randomized controlled trial to determine if a standardized electronic order set, with an embedded computerized decision support system and audit and feedback component, affects the use of appropriate VTE prophylaxis in hospitalized medical patients. The unit of randomization in this study is the hospital, which serves as the cluster. The unit of observation in this study is the individual patient. The primary outcome of this study is the proportion of in-hospital days during which appropriate VTE prophylaxis is administered, in intervention versus control hospitals. Secondary outcomes are the rates of hospital-acquired VTE, major bleeding and mortality, in intervention versus control hospitals. Design, analytic and ethical challenges unique to cluster randomized trials will also be discussed. Strategies to overcome them in this trial will be presented.</p> / Master of Science (MSc)