Targeted Metabolic Profiling in Deep Vein Thrombosis

Deep venous thrombosis (DVT) is a blood clot, usually affecting the legs, causing pain, swelling, and redness. The clot damages the veins, which can result in chronic pain, swelling and ulceration. This is called the post-thrombotic syndrome, which impacts heavily on patients' life and work. If the clot dislodges and travels to the lungs, it becomes a pulmonary embolus (PE), which can be life threatening. Together, DVT and PE affect 500,000 people in Europe every year, representing the most common preventable cause of hospital acquired death. They are expensive conditions not only due to their treatment costs, but also secondary to the resulting loss of work days and productivity.

DVT is diagnosed by clinical examination, risk scoring, usually via the Wells Score, and a blood test called D dimer, a fibrin degradation product. D-dimer is specific but not very sensitive. In other words, when the test is negative, it is unlikely that a DVT is present.

However, many conditions can raise D-dimer levels, making it less useful when positive. Duplex ultrasound can confirm the presence of clot but this can be difficult to visualize or is often not seen at all. The clot can take time to form and patients may not experience symptoms immediately. This is a problem for treatment, as new, clot-busting medication works best in the first 2 weeks after a DVT and it is currently impossible to tell when the thrombus formed.

Because of the importance of DVT, and ongoing issues regarding its diagnosis, ageing and prognosis, numerous research groups have been exploring novel candidate biomarkers to help develop a more accurate diagnostic and prognostic marker for DVT. The Section of Vascular Surgery and of Computational and Systems Medicine (CSM) at Imperial College, have had a longstanding collaboration working towards this end using metabonomics.

Metabonomics is defined as: 'the quantitative measurement of the multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification'. In other words, it examines the end products of cellular metabolism via platforms such as Nuclear Magnetic Resonance Spectroscopy (NMR) and Mass Spectrometry (MS), which are both high throughput analytical platforms. These enable the qualification (untargeted) and quantification (targeted) of different molecules in a given sample, to a degree of detail superior to that offered by other omic technologies (e.g. proteomics). Metabonomics has demonstrated clear applications in vascular disease, including chronic venous disease , atherosclerosis , venous leg ulceration, aneurysmal disease and stroke.

Untargeted preliminary work on deep venous thrombosis has revealed a metabolic signature for DVT using a murine animal model [9], and promising pilot data in a human study. The aim of the proposed study is to validate previous findings in a separate patient cohort and perform quantification of the metabolites identified in the pilot studies via targeted NMR and MS experiments, continuing the exploration of novel biomarkers for DVT.

Study Design

The study is a prospective observational case-control study, recruiting 40 DVT patients and 40 healthy subjects. Patients diagnosed with a DVT will be recruited at the sites of Imperial College Healthcare NHS Trust.

Summary Forty patients diagnosed with a DVT will be recruited. Participation in the study will not interfere with the decision making and diagnostic investigations by the direct care team. A second control group of 40 healthy volunteers will be also examined. Healthy subject recruitment will take place at the same sites. A patient information leaflet will be provided to participants for informed consent purposes.

A member of the research team will explain the study to prospective participants. Both patients and volunteers will be given sufficient time to read the information sheet and make an informed decision regarding participation in the study; this will be confirmed by the participant signing a consent form. The study involves a comprehensive history and clinical examination, recording medication taken and collection of blood and urine samples. The samples will be labeled anonymously with a study number and transferred to the Imperial

College laboratories in South Kensington. Sample transfer will be performed securely according to established departmental standard operating procedures (SOPs). A blood sample will be stored for future DNA analysis in DVT patients to complement the findings from the metabonomic assays. No DNA analysis will be performed for this particular project.

The DVT group will have a repeat ultrasound study, performed by a vascular research fellow on a dedicated (non-NHS) machine 3 weeks after anticoagulation treatment has ended. They will also have repeat urine and blood samples taken. None of the research interventions will have an impact on the clinical care the patients will receive.

A letter explaining the purpose of the study will be sent to the General Practitioner if the participant wishes. All clinical and research information will remain confidential.

Patient Groups:

• Group 1: Patients with DVT confirmed by lower limb venous duplex ultrasound (DUS) (n =40)
• Group 2: Healthy volunteers with no DVT confirmed on DUS (n = 40)