Conventional flow cytometry is a tool used to detect and measure the physical and chemical characteristics of a set of cells. It is very useful for understanding inflammatory profile of diseases. At present, the technique relies on analysis of blood samples using relatively large volumes of blood and it is not suitable for samples derived from children where it is unethical to take such volumes. Thus, inflammatory diseases that are more common in children, such as Immunoglobulin A Vasculitis, suffer from a lack of scientific insight into their pathogenesis. We are looking to optimise the sample preparation technique by proposing the combination of a dielectrophoresis chip and mass cytometry (MC). Dielectrophoresis uses non-uniform electric fields to polarise suspended particles, and Dr. Hoettges has developed a dielectrophoresis chip that efficiently separates leukocytes from red blood cells with minimal cell loss. MC is a novel technology that allows for greater phenotypic resolution than conventional flow cytometry (typically using >45 parameters within a single panel) of immune cell populations therefore providing more efficient use of precious samples. The combination of these techniques may enable analysis of the inflammatory profile in paediatric diseases.
Dielectrophoretic cell separation in combination with mass cytometry will enable phenotypic analysis of lymphocyte cell subsets in children.
1) Optimise dielectrophoresis/MC to determine the smallest volume of blood required to successfully perform lymphocyte profiling.
2) Perform proof-of-concept lymphocyte profiling in paediatric samples obtained from children with inflammatory disease.
- Aim 1:
Method development and optimisation will be performed using adult blood collected from healthy volunteers at Alder Hey Children’s NHS Foundation Trust (approved by the University of Liverpool ethics committee - Research Ethics and Approval Number - RETH000773). Initial work will be performed using conventional flow cytometry and a panel of antibodies. Once robust isolation/staining procedures are in place, MC with a larger panel will be applied and optimised with the dielectrophoresis chip to establish the lowest volume required to obtain sufficient data.
Techniques and training –isolation of leukocytes from peripheral blood samples using conventional and dielectrophoretic techniques and conventional flow cytometry and mass cytometry.
- Aim 2:
As proof-of-concept, a cross-sectional cohort of patients with inflammatory disease, forming part of ‘The Immunoglobulin A Vasculitis (IgAV) Study’ (REC17/NE/0390), will be recruited to take part in this project. A blood sample will be taken from 3 cohorts of patients (n=5 each); (i) ‘IgAV no nephritis’ group – children with IgAV and no nephritis, (ii) ‘IgAV nephritis group’ – children with IgAV with nephritis, (iii) age- and sex-matched paediatric controls from children who attend for blood tests for non-inflammatory reasons. The samples will be processed using the protocols in Aim 1 and leucocyte subset comparisons made between the groups using appropriate analytical software.
Techniques and training – aware of consenting and recruiting patients for translational research, patient sample processing, analysis of large data sets using appropriate statistical and analysis software.
This project will provide evidence as to whether small volumes of blood can be used for lymphocyte analysis, enabling a novel technique for paediatric inflammatory research.
- Dr Louise Oni and Dr Rachael Wright (Experimental Arthritis Treatment Centre for Children) - http://www.eatc4children.co.uk https://www.liverpool.ac.uk/translational-medicine/staff/louise-oni/
- Dr Joseph Slupsky (Molecular and Cellular Cancer Medicine) - https://www.liverpool.ac.uk/translational-medicine/staff/joseph-slupsky/
- Dr Kai Hoettges (Department of Electrical Engineering and Electronics) – https://www.liverpool.ac.uk/electrical-engineering-and-electronics/staff/kai-hoettges/
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/