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Title: Plasma microparticles in patients with gynaecological malignancy
Author: Zahra, Sharon
ISNI:       0000 0004 6425 3728
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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Gynaecological malignancy is a common diagnosis that is highly amenable to effective treatment. Unfortunately the incidence of thrombosis, together with its morbidity and mortality, remains significant in this patient group. Patients with thrombosis have also been shown to have a worse prognosis. Thromboprophylaxis can reduce the incidence of thrombosis and may potentially also improve outcomes. Primary thromboprophylaxis could be indicated in patients considered to be at high risk of thrombosis. However empirical thromboprophylaxis is not without its risks as cancer patients also have a significant risk of bleeding. Primary thromboprophylaxis cannot at present be recommended for all patients with gynaecological malignancy. We are currently unable to assess whether or when a thrombosis is likely to happen. The ability to identify patients at a high risk of thrombosis would allow a more tailored approach to primary thromboprophylaxis that could translate into improved outcomes. Microparticles are postulated to be important participants in many pathophysiological processes, including haemostasis and thrombosis. There is also increasing evidence that microparticles influence cancer cell survival, invasiveness and metastasis. There are several publications in peer reviewed literature reporting increased numbers of circulating plasma microparticles in patients with malignancy and other prothrombotic conditions. It is hypothesised that microparticles may be directly involved in the pathogenesis of thrombosis and in cancer cell survival and progression, and hence may have a direct effect on overall prognosis and outcome. Measuring the number of circulating plasma microparticles could potentially help identify cancer patients at increased risk of thrombosis that may benefit from primary thromboprophylaxis, which in turn could potentially lead to improved overall outcomes. I set out to quantify and compare the number of circulating plasma microparticles in patients with gynaecological malignancy and a control group of women with no malignancy. I also wanted to establish whether measuring the number of plasma microparticles would identify patients at an increased risk of thrombosis. Research on microparticles is currently hampered by the lack of a standardised method for the identification and quantification of microparticles, mainly due to their small size. There are several pre-analytical variables that may influence the number of microparticles identified. These include the isolation methods, the labelling antibodies and the assays used to quantify microparticles, amongst others. Use of different laboratory techniques by different investigators makes the meaningful comparison of results in the published literature difficult. Flow cytometry is the technique most commonly used to quantify microparticles. I set up a laboratory assay based on light-scattering flow cytometry to identify and quantify microparticles. Platelet free plasma was prepared from citrated blood, monoclonal antibodies were used to label microparticles, and a flow based method used to quantify the absolute number of microparticles in the patient plasma by flow cytometry. There is currently still debate as to whether microparticle assays need to be done on fresh plasma samples or whether they can be meaningfully done on frozen samples. In the first twenty patients I compared the results obtained from fresh plasma with the results from a separate aliquot of the same plasma sample that had been stored frozen at -80°C. The number of microparticles detected after a freeze/thaw cycle did not correlate with the results obtained from fresh plasma. In view of this I analysed all patient samples on fresh plasma within two hours of collection. All patient samples in my study have been handled in an identical manner to ensure that results from different patients are comparable. I recruited 67 women with gynaecological malignancy and a control group of 42 women without malignancy. I measured the total number of circulating plateletderived microparticles, leucocyte-derived microparticles, endothelial-cell derived microparticles, tissue factor positive microparticles and annexin V positive microparticles. There was no statistically significant difference in the number of circulating microparticles between the patients with gynaecological malignancy and the women without malignancy. Five (7.5%) of the patients with gynaecological malignancy were diagnosed with a venous thrombotic event during the study follow up period (median of 11 months, range 5-17 months). The number of plasma microparticles in the latter patients was indistinguishable from that in gynaecological cancer patients without thrombosis and in the control group. However, because only a small number of patients (five) were diagnosed with a thrombosis, it has not been possible to ascertain whether such patients truly do not have raised levels of microparticles, or whether a larger study might demonstrate a difference. In order to establish whether my study cohort is representative of gynae-oncology patients in the South East of Scotland Cancer Network (SCAN), I identified all patients diagnosed with a gynaecological malignancy in SCAN during 2009 (period of study recruitment). I compared the characteristics of this group of patients with my study cohort and found that my study cohort was highly representative of the overall population of patients with gynaecological malignancy in SCAN with a similar frequency of the different gynaecological malignancies and a similar incidence of thrombosis. There are a number of limitations to this study. There is still no universally accepted definition of what constitutes a microparticle. The assay 1 have used, while standardised for this study, is not universally used by all investigators measuring microparticles. Although flow cytometry is the commonest technique used to measure microparticles, the accuracy of the assay is called into question given the small size of the microparticles being measured. Further this is a single centre study, and although all the patients recruited had been diagnosed with gynaecological malignancy, given the variable nature and prognosis of the different types of gynaecological cancers, the patients recruited are in fact a diverse group. Recruiting patients with a specific type of gynaecological cancer only would have led to a more uniform group of patients that are more readily comparable. The overall number of patients diagnosed with a venous thromboembolic event, while representative of the overall population in SCAN diagnosed with gynaecological cancer, is too small to be able to draw any firm conclusions regarding the usefulness of measuring microparticles as a prothrombotic marker or otherwise, particularly given the different incidence of venous thrombotic events in the different types of gynaecological cancer. As far as I can establish this is the first study looking at microparticles specifically in patients with gynaecological malignancy. Keeping the limitations of the study in mind, I did not find a difference in the number of plasma microparticles in patients with gynaecological malignancy compared to a control group of women with no cancer. Further, microparticles cannot at present be used to help identify patients with gynaecological malignancy at a higher risk of venous thrombotic events.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available