MMed, Haematology. University of the Witwatersrand, Faculty of Health Sciences / Thrombocytopenia is common among individuals infected with HIV, with a wide range of possible causes. It often necessitates the performance of a bone marrow investigation in order to assess megakaryocyte activity and to exclude the presence of bone marrow infiltration. Unfortunately, meaningful interpretation of the bone marrow findings is often hampered by the frequent co-existence of multiple potential pathogenic processes. For example, megakaryocyte numbers are often well preserved (even in the presence of marrow infiltration), but show a degree of dysplasia, (suggesting that ineffective megakaryopoiesis is contributing to the thrombocytopenia). In addition, processes associated with peripheral platelet consumption (such as immune-mediated platelet destruction or disseminated intravascular coagulation) are also common, and the mechanism causing thrombocytopenia is therefore often obscure. Because this mechanism is of clinical interest, (in that it guides the selection of the most appropriate therapy), a functional test of megakaryocyte activity would be of potential value. The IPF is a platelet parameter measured on the Sysmex XE-5000 haematology analyzer, which quantifies the number of reticulated platelets, and has been shown to be a good reflector of underlying bone marrow megakaryocyte activity. The objectives of this study were therefore to measure the IPF level in HIV-positive patients with thrombocytopenia who had undergone a bone marrow investigation, and to correlate the IPF with the bone marrow morphology findings and other clinical variables of interest (including the CD4 count, the HIV viral load and the presence of opportunistic infections or malignancies). The IPF was also assessed as a tool to predict the short term platelet count response to the therapy initiated by the attending clinician. 78 patients were enrolled, of whom 38 (49%) had mycobacterial infection, 12 (15%) had ITP/TTP and eight (10.3%) had a malignancy. CD4 counts were available in 70 patients, of whom 63 (90%) had AIDS. Thirty seven patients (47%) were found to have an IPF level greater than 7.7% , with an overall population mean IPF level of 9.5%. A strong relationship was identified between the IPF and the platelet count, with 81% of patients with grade four thrombocytopenia having an IPF level greater than 7.7% (mean IPF=14%), as compared to only 8% of grade one thrombocytopenia (mean IPF=6%). 67% of patients with hypocellular or extensively infiltrated marrow had a low IPF (≤ 7.7%)(mean=7%), as compared to only 25% of patients with ITP/TTP (mean=14.8%). A higher proportion of patients with low viral load levels had a low IPF as compared to those with higher viral loads, possibly due to the apparent sparing of patients with low viral loads from grade four thrombocytopenia. In contrast, the presence of a significant degree of megakaryocyte dysplasia, a very low CD4 count or the presence of mycobacterial infection did not affect the IPF distribution, suggesting that the underlying mechanism causing thrombocytopenia in these subgroups was heterogeneous. An IPF level greater than 10% predicted a partial platelet count response (as defined as an improvement by greater than 50% of the baseline platelet count to a minimum level of 20x10^9/l), with a specificity of 81% and a positive predictive value of 79%, while an IPF level less than 6% had a specificity of 89% and a positive predictive value of 70% for a complete failure to show a platelet count response to the therapy instituted. Limitations of the IPF illuminated in this study include a loss of reliability in any circumstance in which the platelet count as measured by optical fluorescence may be in question, and includes disorders associated with profound red cell fragmentation and some malignancies. The cause of thrombocytopenia is concluded to be very heterogeneous in thrombocytopenic patients with AIDS, even among patients with a unifying diagnosis (such as mycobacterial infection). The IPF is therefore a useful tool to assist the morphologist in interpreting the bone marrow findings in this clinical setting, as well as in predicting the short term platelet count response to therapy.

Les thrombopénies constitutionnelles (TC) sont des maladies encore incomplètement caractérisées. Ce travail de thèse concerne les TC liées à 10 variants des gènes codant les facteurs de transcription FLI1, ETV6 et GATA1, dont 9 n’avaient jamais été décrits. Ces pathologies ont été étudiés à partir de patients recruté dans un réseau national (Centre de Référence des Pathologies Plaquettaires CRPP) et un réseau international (BRIDGE consortium).Nous montrons qu’il existe un déficit sévère en granules denses dans les plaquettes des patients porteurs de variants FLI1 du fait d’un probable défaut de biogénèse. Ce travail, et d’autres études publiées récemment, ont permis de définir la TC liée aux variants ETV6 en tant que nouveau syndrome de prédisposition aux hémopathies malignes. Les variants FLI1 s’associent à une diminution d’activité transcriptionnelle et d’accumulation nucléaire de la protéine et à des anomalies de la différenciation mégacaryocytaire. Les variants ETV6 s’associent à un défaut d’activité répressive et les mégacaryocytes dérivés des patients montrent un excès de prolifération et un défaut marqué de formation des proplaquettes. Les plaquettes de patients porteurs de variants GATA1 ont montré une expression anormale de la protéine MYH10, ce qui suggère un défaut de répression du gène MYH10 au cours de la mégacaryopoïèse. Une analyse in silico de données de ChIP-seq a ainsi montré l’existence d’une fixation de GATA1 dans le promoteur et dans un intron de MYH10 dans le mégacaryocyte.Ce projet a permis d'apporter des connaissances sur les causes génétiques, le phénotype, le diagnostic, le pronostic et les mécanismes physiopathologiques des TC. / Constitutional thrombocytopenia (CT) is a group of diseases incompletely characterized. This thesis focused on CTs due to 10 variants in genes encoding the transcription factors FLI1, ETV6 and GATA1, of which 9 had never been described. These diseases were studied in French and European patients recruited using national (French national reference center for inherited platelet disorders CRPP) and international (BRIDGE consortium) networks.We showed that the platelets of patients carrying FLI1 variants harbored a severe dense granule defect probably due a biogenesis defect. Our work, associated with data published by other groups, has defined ETV6-related CT as a new hematological malignancy predisposition syndrome. FLI1 variants are associated with a decreased transcriptional activity, a decreased nuclear accumulation of the protein and abnormal megakaryocyte differentiation. ETV6 variants led to a decreased repressive activity and the megakaryocytes derived from patients showed increased proliferation and a marked defect in proplatelet formation. The platelets of GATA1 variant carriers showed aberrant expression of MYH10 protein suggesting a defective silencing of MYH10 gene during megakaryopoiesis. Consistently, in silico analysis of ChIP-seq data showed that GATA1 binds the promoter and an intronic region of the MYH10 in megakaryocytes.This project has provided insights into genetic causes, phenotype, diagnosis, prognosis and pathophysiological mechanisms of CTs.

No description available.

Background: Thrombocytopenia is common in sick preterm babies in the first day of life. Despite this, platelet production in thrombocytopenic preterm babies has rarely been assessed. Methods: To address this problem I have developed miniaturised assays to study circulating megakaryocyte (MK) progenitors (BFU-MK and CFU-MK), total cultured MK precursors and mature MK, by culturing mononuclear cells purified from 0.5-1ml of preterm peripheral blood. MK lineage colonies and cells are identified by a MK specific anti-glycoprotein IIb/IIIa antibody (CD61) by APAAP. Results: i) Normal values for these cells at birth were established in cord blood from healthy term and preterm babies. ii) Circulating BFU-MK/CFU-MK, total cultured MK precursors and mature MK were then prospectively studied in 63 preterm babies (gestational age 24-34 wks). iii) At birth 18 (69%) of the thromocytopenic babies were also neutropenic. Conclusion: These data indicate that the principal cause of the thrombocytopenia and neutropenia in the preterm babies studied was <I>reduced platelet</I> <I>and neutrophil production</I> occurring as a consequence of reduced numbers of MK and neutrophil progenitors respectively. Taken together these data suggest the haematological abnormalities characteristic of new-born born to mothers with PIH or with IUGR (thrombocytopenia, neutropenia and polycythaemia) are a consequence of dysregulation of <I>fetal haemopoiesis</I> occurring proximal to committed MK and neutrophil progenitors, most likely at the level of the primitive multipotent haemopoietic stem cell (CFU-GEMM). Finally the value of miniaturised MK progenitor and precursor assays in evaluating rare or unusual cases of neonatal thrombocytopenia has been demonstrated. In the three examples investigated this approach provided unique insights into the pathogenesis of the thromocytopenia in each case.

Cyclical thrombocytopenia (CT) is a rare hematological disease characterized by periodic oscillations in the platelet count. Although first reported in 1936, the pathogenesis and an effective therapy remain to be identified. Since besides fluctuations in platelet levels the patients hematological profile have been consistently normal, a destabilization of a peripheral control mechanism might play an important role in the genesis of this disorder. In this thesis, we investigate through computer simulations the mechanisms underlying the platelet oscillations observed in CT. First, we collected the data published in the last 40 years and quantified the significance of the platelet fluctuations using Lomb-Scargle periodograms. Our analysis reveals that the incidence of the statistically significant periodic data is equally distributed in men and women. The mathematical model proposed in this thesis captures the essential features of hematopoiesis and successfully duplicates the characteristics of CT. With the same parameter changes, the model is able to fit the platelet counts and to qualitatively reproduce the TPO oscillations (when data is available). Our results indicate that a variation in the megakaryocyte maturity, a slower relative growth rate of megakaryocytes, as well as an increased random destruction of platelets are the critical elements generating the platelet oscillations in CT. / La thrombocytopénie cyclique (TC) est une rare maladie hématologique caracteriséepar des oscillations périodiques dans les plaquettes sanguines. Bien qu'elle fût évoquéepour la première fois en 1936, la maladie et une thérapie efficace restent à trouver.Puisque malgré les fluctuations au niveau des plaquettes, les profiles hématologiquesdes patients restent toujour normaux, une destabilisation du méchanisme de contrôlepériphérique peut jouer un rôle important dans la formation de ce maladie. Dans cettethèse, nous recherchons à travers des simulations informatiques les mechanismes sousjacentaux oscillations des plaquettes observées dans TC. En premier lieu, nous avonscollecté les données publiées ces 40 dernière années et quantifié l'importance des fluctuationsdes plaquettes en utilisant les périodograms Lomb-Scargle. Notre analysestatistique révèle que les données périodiques sont équitablement distribuée chez leshommes et les femmes. Le modèle mathématique proposé dans cette thèse prenden compte les caractéristiques essentielles de la production des cellules sanguineset reproduit avec succès les charactéristiques de TC. Avec les même changementde parametèrs, le modèle reproduit bien le comportement des plaquettes sanguineset donne qualitativement les même oscillations que TPO (quand les données sontdisponibles). Nos résultats indiquent que les éléments critiques générant les oscillationsdes plaquettes dans TC sont une variation dans la maturité du mégakaryocytes,un taux de croissance relativement lent des mégakaryo cytes , ainsi que une augmentationaléatoire de destruction des plaquettes.

Thesis (M.D.) -- Dept. of Medicine, University of Adelaide, 1974.

No description available.

The use of an appropriate anticoagulation regiment for the treatment of venous thromboembolism (VTE) in patients with concomitant thrombocytopenia is based on anecdotal evidence and the opinions of managing physicians. The current guidelines suggest that therapeutic levels of anticoagulants may be safely administered to patients who have a minimum platelet count of 50 x 109/L. However, it has recently been suggested that the minimal platelet threshold for safe anticoagulation treatment can be provided at a reduced platelet count of 30 x 109/L. Thus, in order evaluate these platelet threshold we used a thromboelastography (TEG) model to evaluate the clotting parameters of whole blood at predefined platelet counts in the presence of unfractionated heparin (UFH) and low-molecular weight heparin (LMWH). Due to the importance of red blood cells on hemostasis a whole blood TEG model was designed in order to mimic in vivo hemostasis. Clotting was initiated using different concentrations of tissue factor for each anticoagulant at therapeutic and prophylactic levels of UFH and LMWH at predefined platelet counts. In the presence of therapeutic concentrations of either UFH or LMWH, there were no significant differences in TEG parameters of whole blood clots between platelet counts of 30 x 109/L and 50 x 109/L when clotting was driven by the extrinsic pathway. At prophylactic levels of LMWH clot formation was less compromised. Furthermore, no significant difference was noted between platelet-depleted blood (PDB; <10 x 109/L) and 30 x 109/L with respect to r-time. This suggests LMWH at prophylactic levels has no significant bearing on clot formation at a lower platelet threshold versus therapeutic levels of LMWH. Overall, it shows that clot formation is similar for UFH and LMWH when platelet counts are reduced from 50 x 109/L to 30 x 109/L. This work provides insight on the potential for anticoagulation at a reduced platelet threshold in thrombocytopenic conditions. / 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.

No description available.