Research and Development
Triennial Report 2009-12
Cover Image.
(Left) An immature reticulocyte, caught in the act of enucleation. The cell was cultured, ex vivo, from adult peripheral blood
CD34+ progenitor cells. Copyright statement. This research was originally published in Blood. Griffiths RE, Kupzig S, Cogan N,
Mankelow TJ, Betin VM, Trakarnsanga K, Massey EJ, Lane JD, Parsons SF, Anstee DJ. Maturing reticulocytes internalize plasma
membrane in glycophorin A – containing vesicles that fuse with autophagosomes before exocytosis. Blood. 2012; 119:6296-306.
© the American Society of Hematology. Pseudocolored scanning electron micrograph courtesy of Sabine Kupzig.
See the article by David Anstee on page 31.
(Right) Megakaryocytes grown in vitro from induced pluripotent stem cells. See the article by Dr Cedric Ghevaert on page 42.
Research and Development Triennial Report 2009-12
Contents
Foreword...............................................................................................................................5
Introduction..........................................................................................................................6
Theme 1 – Donor health and behaviour
INTERVAL study, Professor John Danesh and Professor David Roberts ................................................. 10
Cambridge BioResource and National Institute for Health Research (NIHR)
BioResource for Rare Diseases, Dr Nicola Foad, Dr Sarah Morley, Dr Jennifer Sambrook....................... 11
Theme 2 – Transfusion/transplantation virology and microbiology
Transfusion microbiology with respect to the microbiological safety of blood, tissues and organs,
Professor Richard Tedder.................................................................................................................... 12
Virological blood safety, Professor Jean-Pierre Allain........................................................................... 13
Reactivation of common viruses in patients receiving chemotherapy or immunosuppressive drugs,
Dr Daniel Candotti............................................................................................................................. 14
Blood donors, viral infections and malaria in Ghana, Professor Jean-Pierre Allain................................. 15
A screening test for anti-malarial antibodies, Professor David Roberts.................................................. 16
Non-coding RNAs in cytomegalovirus infection, Dr Lars Dölken ......................................................... 17
Transfusion medicine epidemiology review: TMER, Dr Patricia Hewitt.................................................. 19
Screening assays for Prion disease, Professor David Anstee ................................................................. 20
Theme 3 – Appropriate and safe use of blood components
Transfusion alternatives before surgery in Sickle Cell Disease (TAPS), Dr Lorna Williamson.................... 21
Studies into the clinical effectiveness of platelet transfusions and the optimal product,
Dr Simon J Stanworth........................................................................................................................22
Use of information technology to deliver safer and more effective transfusion practice,
Professor Mike Murphy......................................................................................................................23
Systematic Review Initiative (SRI), Professor Mike Murphy................................................................... 24
NHSBT/Medical Research Council – Clinical Studies Unit, Dr Charlotte Llewelyn................................... 25
Components Development Laboratory, Dr Rebecca Cardigan.............................................................. 27
Theme 4 – Erythrocyte biology and immunology
Making and manipulating red blood cells in health and disease, Dr Ashley Toye.................................. 29
Structure and function of red blood cells, Professor David Anstee....................................................... 31
Erythropoiesis in health and disease, Professor David Roberts.............................................................. 32
Red cell diagnostics, Professor Marion Scott.......................................................................................34
Research and Development Triennial Report 2009-12
3
Contents
Theme 5 – Platelet biology and genomics
Placing signposts in the genome for the formation and function of platelets,
Professor Willem Ouwehand..............................................................................................................36
Functional genomics interpretation of the genetic determinants of haematological parameters,
Dr Augusto Rendon........................................................................................................................... 37
A blueprint of haematopoietic epigenomes, Professor Willem Ouwehand...........................................39
Elucidating the function of novel genes in haematopoiesis in zebrafish, Dr Ana Cvejic.........................40
Platelet biology and quantitative trait loci, Stephen Garner and Dr Peter Smethurst............................. 41
In Vitro production of platelets for transfusion, Dr Cedric Ghevaert..................................................... 42
A study of the HPA-1a Antibody Response in Neonatal Alloimmune Thrombocytopenia (NAIT),
Professor David Roberts...................................................................................................................... 43
Recombinant antibody for treatment of fetomaternal alloimmune thrombocytopenia,
Dr Cedric Ghevaert............................................................................................................................44
Theme 6 – Organ donation and transplantation
Quality in Organ Donation (QUOD), Professor Rutger J Ploeg.............................................................. 45
Impact of donor factors on transplant outcome, Professor Dave Collett...............................................46
Allocation of organs for transplantation, Rachel Johnson.................................................................... 47
Cancer in transplant recipients, Professor James Neuberger.................................................................48
Theme 7 – Stem cells and immunotherapies
Immunogenetic markers in transfusion and transplantation,
Dr Cristina Navarrete and Dr John Girdlestone....................................................................................49
Immunoregulatory properties of cells derived from umbilical cord and cord blood,
Dr Cristina Navarrete and Dr John Girdlestone....................................................................................50
Improving cord blood transplant outcomes by understanding the bone marrow niche,
Professor Suzanne Watt..................................................................................................................... 52
Stem cell therapies for heart disease, Dr Enca Martin-Rendon and Professor Suzanne M Watt.............54
Regulatory T-cell therapy for graft verse host disease following human stem cell transplantation,
Dr Wei Zhang, Professor Suzanne Watt and Professor David Roberts................................................... 55
Developing antigen-specific adoptive cellular immunotherapy for CMV and EBV, Dr Frederick Chen....56
Stem cells in leukaemia, Dr Allison Blair.............................................................................................. 57
Theme 8 – Molecular and tissue engineering
Development of 2nd and 3rd generation tissue grafts, Professor John Kearney....................................58
Tissue Development Laboratory – from research to service, Dr Paul Rooney......................................... 59
Creating new blood vessels for tissue repair, Professor Suzanne Watt..................................................60
The work of the Clinical Biotechnology Centre in supporting molecular therapy trials,
Dr Paul Lloyd-Evans............................................................................................................................ 61
Awards and honours 2009-11............................................................................................62
Publications 2009-11...........................................................................................................63
Patents 2009-12..................................................................................................................73
4
Research and Development Triennial Report 2009-12
Foreword
I have been delighted to
chair NHSBT’s Research and
Development (R&D) Committee
for the past three years. There is
no doubt that both research and
development are fundamental
to NHSBT’s overall status as
a forward-looking, flexible
organisation.
An active R&D programme maintains credibility with
customers, contributes significantly to our highly rated
international reputation for scientific excellence and enables
us to attract medical and scientific talent. All of which
benefits both teaching and service provision.
The last three years saw further evolution of NHSBT’s research
programme, to ensure across-the-board underpinning of
the organisation’s objectives in organs, blood, stem cells and
tissues, while maintaining high scientific quality.
In 2010, international experts undertook the quinquennial
scientific peer review of our research programmes. I am
delighted to report that this concluded that the research
programmes led by our PIs are world class in the areas of
erythrocyte biology, virology, cellular engineering, clinical
studies and platelet genomics. In addition, translational
research was considered strong in the areas of tissues and red
cell diagnostics.
As always, the challenge is to ensure that research and
routine services are appropriately connected with each other.
In order to achieve this, we have established eight strategy
groups which link research, development and operations.
Their influence in shaping our research programme will
undoubtedly grow in the coming years.
I am very pleased that we have launched two new coordinated research programmes. The first is in the area of
donor health and behaviour and the second is in organ
donation and transplantation.
With a predicted increase in demand for blood over the next
decade, it is critical that we avoid detriment to donors’ health
and wellbeing, while maximising donation opportunities.
The new programme in donor health and behaviour aims to
achieve both these goals. Our donors have also contributed
to NHS biobanking activities, thus extending their gift to
research into common diseases such as diabetes and coronary
artery occlusion. In organ donation and transplantation, we
will create a UK-wide organ biobank to underpin vital studies
to improve the number and quality of organs for donation.
Remembering that three patients die each day in the UK
from lack of an organ, research to predict which organs can
be used is essential, along with strategies to maintain their
viability between retrieval and transplantation.
It is, of course, critical to ensure that our research strength
continues into the future. The recruitment of five new
Principal Investigators and a strong programme for trainees
and clinical fellows has injected new vigour into the team.
Despite financial pressures, our researchers have continued to
attract an excellent level of external funding, and their efforts
maintain NHSBT as a leading scientific blood and transplant
organisation. From molecular diagnostics to manufacture
of blood cells from stem cells, our research programme
continues to provide translation of science into new products
and services.
Finally, I would very much like to thank the external
international experts on our R&D committee, Professors
Harvey Klein, Lucio Luzzatto and Rutger Ploeg, who give
up so much time in diligent preparation and whose advice
is invaluable in our strategic decision making. This Triennial
Report demonstrates the range and depth of NHSBT’s
research programme, which bodes well for the organisation’s
future.
Bill Fullagar
Chair, NHSBT
Chair of R&D Committee.
Research and Development Triennial Report 2009-12
5
Introduction
The last three years has been an
exciting time for Research and
Development (R&D), with renewal
of funding from the National
Institute of Health Research
(NIHR), scientific and structural
reviews leading to Board approval
of a reshaped four-year research
strategy and recruitment of a new
cadre of Principal Investigators (PIs).
Advances in molecular diagnostics
and generation of blood cells from stem cells have moved
apace, and we have also launched new strategic initiatives in
donor population health (INTERVAL trial) and organ transplant
biobanking to support research aimed at improving organ
numbers and quality (QUOD programme).
Research across NHSBT now involves 17 PIs, close to 200 staff
and a total budget of – £16M/yr. The main research sites, in
collaboration with University partners, are Cambridge, Oxford,
London and Bristol, with specialist activity in Birmingham and
Liverpool. Our supporting infrastructure consists of the Clinical
Biotechnology Centre (Bristol), which manufactures GMP-grade
biologicals for Phase 1 clinical trials, the NHSBT/MRC Clinical
Studies Unit (Oxford/Cambridge/London), which supports
clinical studies and trials, the Systematic Reviews Initiative
(Oxford) and the Statistics and Clinical Audit team (Bristol).
NIHR programmes
In late 2009, NIHR invited us to rebid for a new five-year cycle
of funding, superseding the funding we already received.
In a very short space of time, four high quality programmes
were submitted to a total value of £14M over five years, and
funding was approved in full for Programmes B-D. Since
Programme A was new and innovative, NIHR asked for an
18 month checkpoint report. Just before this Triennial Report
went to press, we heard the excellent news that funding
has now been approved in full for the remainder of this
programme. The individual programmes are:
• Programme A: Molecular and Tissue Engineering.
Co-ordinator Professor Amit Nathwani
• Programme B: Megakaryocyte and Platelet Biology and
Genomics. Co-ordinator Professor Willem Ouwehand
• Programme C: Stem Cell and Immunotherapies: Improving
Bone Marrow Transplant Outcomes.
Co-ordinator Professor Suzanne Watt, and,
• Programme D: Erythropoiesis in Health and Disease.
Co-ordinator: Dr David Roberts.
These programmes form a major proportion of NHSBT’s
laboratory research.
Scientific and structural reviews
In late 2010, our entire R&D programme underwent its
scientific quinquennial review. Each major site was visited by
a team of international reviewers who met with PIs, scientists
and PhD students, the latter to discuss training. I am very
grateful to the reviewers who gave up so much time to help
us with these reviews, especially as two of them were then
stuck in the UK for days afterwards by snow at Heathrow!
6
Research and Development Triennial Report 2009-12
The review teams were:
• Cambridge: Professor Harvey Klein, Professor Kenneth
Clemetson
• Filton: Professor Lucio Luzzatto, Professor Martin Olsson
• Oxford: Professor Mo Blajchman, Professor Jeff
McCullough, and
• ODT: Professor Rutger Ploeg, Professor Anthony Hollander.
The main conclusions were that the research programmes
were world class in the areas of erythrocyte biology,
virology, cellular engineering, clinical studies and platelet
genomics, and that translational research is strong in the
areas of tissues and red cell diagnostics. Research in organ
transplantation was strong, but was considered to require
greater resources and better co-ordination in order to achieve
a quantum leap in output and translation. This and other very
helpful recommendations have been crucial in reshaping the
R&D programme.
A parallel review of the organisation of research and its links
with the rest of NHSBT’s activities concluded that connections
between research, development and operational business
were variable, and could be strengthened in all areas. In
addition, it was considered that accountability for progress
on research projects could be more streamlined, and that the
infrastructure to support R&D needed to be strengthened to
support the expansion in activity and complexity of regulation.
New research strategy
Taking the recommendations from the reviews into account,
in early 2011, NHSBT’s Board approved a new four-year
research strategy, which organised research into eight
themes linked to areas of business. The intention is that the
programme will be increasingly developed by a strategy
group for each theme, comprising research, development and
operational staff.
The research themes approved in the 2011 strategy are:
1. Donor health and behaviour
2. Transfusion and transplantation virology and
microbiology
3. Appropriate and safe use of blood components
4. Erythrocyte (red cell) biology and immunology
5. Platelet biology and genomics
6. Organ donation and transplantation
7. Stem cells and immunotherapies, and
8. Molecular and tissue engineering.
Most of these covered work already in place, but two new
themes were added:
Theme 1: (Donor health and behaviour)
In collaboration with the University of Cambridge Department
of Public Health and Primary care (Professor John Danesh),
we have commenced a large strategic study (INTERVAL),
which aims to provide evidence to determine policy on the
optimal interval between blood donations. This study seeks
to minimise the risk of iron deficiency/anaemia in donors
while maximising the use of a diminishing donor pool. This
ambitious study also has the potential to personalise the
donor call up, by using genetic and other factors to predict
tolerance to donation.
Introduction
R&D Income 2012-13
A new PI, Dr Emanuele di Angelantonio, has just been
appointed to lead future development of this theme.
Figure 1. R&D Income 2012-13
Theme 6: (Organ donation and transplantation)
Although NHSBT has funded transplant projects since its
inception in 2005, these were usually single centre, and
focussed on management of the patient after transplantation.
We have now reoriented this theme to reflect NHSBT’s
responsibility for provision of high quality organs to the NHS.
We have appointed a PI, Professor Rutger Ploeg, previously an
international member of our R&D Committee, and now based
in Oxford to lead this endeavour. In late 2011, funding was
approved to establish an organ biobank to facilitate studies to
improve organ quality.
Blood Price Levy
11%
Department of
Health
3%
External
Income from research
Grant in Aid
Department of Health
Blood Price Levy
External
51%
Grant in Aid
25%
Income from
research
10%
A future aim is to explore partnerships to develop behavioural
studies to promote increased blood and organ donation.
Congratulations to Professor Willem Ouwehand who received
a well-deserved Personal Chair at the University of Cambridge.
Staff organisation and development
We also said goodbye to two PIs who have retired: Professor
Jean-Pierre Allain from Cambridge, and Dr Derwood
Pamphilon from Bristol, both of whom contributed hugely
over many years, and who will be much missed. Jean-Pierre
Allain is an international leader in the field of transfusion
virology, and his strong links with Ghana and other groups
world-wide have provided new insights into the behaviour
and spread of blood-borne viruses. His ‘retirement’ is likely to
be as busy and productive as ever!
NHSBT’s PIs are either clinicians (11) or scientists (6), and all
hold either substantive or honorary University appointments.
Medically qualified PIs are Honorary Consultants within NHSBT,
and three scientist PIs also have high-level service responsibilities.
We have strengthened the programme in the last three years
with the appointment of five new NHSBT’s PIs in the areas of
blood donor population health (Emanuele di Angelantonio),
appropriate blood use (Simon Stanworth), virology (Lars
Dölken), erythrocyte biology (Ashley Toye), and organ donation
and transplantation (Rutger Ploeg). The PIs, the themes in which
they work and their locations are shown in Table 1.
Derwood Pamphilon’s work encompassed many facets.
He was co-leader of the Cellular Therapies team in the
international Biomedical Excellence for Safer Transfusion
Table 1 – The Pls, the themes in which they work and their locations
Theme
Oxford
Cambridge
1. Donor health and
behaviour
Dr Emanuele di
Angelantonio (new 2012)
2. Transfusion and
transplantation virology
and microbiology
Dr Lars Dölken
(new 2011)
3. Appropriate and
safe use of blood
components
Prof Mike Murphy
Dr Simon Stanworth
(new 2010)
4. Erythrocyte biology
and immunology
Prof David Roberts
5. Platelet biology and
genomics
Other
Prof Richard Tedder
(Colindale/UCL)
Prof David Anstee
Prof Marion Scott
Dr Ashley Toye
(new 2010)
Prof Willem Ouwehand
Dr Cedric Ghevaert
(new 2010)
6. Organ donation and
transplantation
Prof Rutger Ploeg
(new 2011)
7. Stem cells and
immunotherapies
Prof Suzanne Watt
8. Molecular and tissue
engineering
Bristol
Chair of transfusion
medicine (vacant)
Dr Allison Blair*
Dr Cristina Navarrete
(Colindale/UCL)
Prof Amit Nathwani
(UCL)
Prof John Kearney
(Liverpool)
Research and Development Triennial Report 2009-12
7
Introduction
(BEST) collaborative, the Joint Accreditation Committee of the
International Society for Cellular Therapy and European Group
for Blood and Marrow Transplantation (JACIE). He leaves a
wonderful legacy of his career as co-author of the awardwinning textbook ‘Practical Transfusion Medicine’, the fourth
edition of which is in preparation.
Budget from within NHSBT consists of £1.6M from blood
price, which is used to fund initiatives in i) Donor health and
behaviour, ii) Transfusion and transplantation virology and
microbiology and, iii) Appropriate and safe use of blood
components. There is also an ODT contribution of 100k
(Theme 6). See Figure 1.
To develop the next generation of talented researchers,
funding has been ear-marked for eight clinical fellow posts to
undertake two or three years full-time research. In addition,
we receive funding from NIHR for post-graduate medical
trainees who wish to follow an academic career. This allows
them time for exposure to research in parallel with their
clinical training, and the opportunity to undertake a fulltime
PhD. We currently have five such trainees in Oxford and
Cambridge, four of them are in haematology and one on an
emergency medicine/anaesthetics care programme. Others
are planned in virology and public health.
PIs and senior scientists are also expected to raise funds from
competitive external sources (industry, research councils,
EU, NIHR competitive funding, charities). Although amounts
available from external funders have reduced, the current
figure is about £6M/yr. Most of this is run through partner
universities, and can be used for more ‘blue sky’ research in
support of the overall strategy.
We have recently initiated a series of development days for
PIs and their team-leaders. As well as sessions on Intellectual
Property and research governance, team exercises aim to
develop management skills to complement scientific expertise.
Funding
Research funding for this period was approximately £16M per
year, comprising:
Funding from the NHS of about £10.2M, derived as
follows:
• National Institute for Health Research: Four Programme
Grants for five years (2010-15), totalling £2.8M/year
• National Institute for Health Research: funding for salaries
of NHS staff supporting research: £900k/year
• National Institute for Health Research: (Flexibility and
Sustainability Funding). Variable amounts are awarded
annually in April using a formula, which takes into account
external funding. Over the last three years, amounts have
varied between £1.8M and £1.32M
• Grant-in-Aid to Bristol’s International Blood Group
Reference Laboratory/Bristol Institute of Transfusion
Science £2.5M, and,
• Income from research products, cell lines and licences
£1M.
R&D Expenditure Allocation by Theme 2012-13
Figure 2. R&D Expenditure allocation by theme 2012-13
Theme 8
18%
Theme 1
7%
Theme 2
10%
Theme 3
4%
Theme 7
13%
Theme 6
5%
Theme 5
9%
8
Theme
Theme
Theme
Theme
Theme
Theme
Theme
Theme
1
2
3
4
5
6
7
8
Theme 4
34%
Research and Development Triennial Report 2009-12
Allocation of funding to the eight research themes is shown
in Figure 2.
Governance
Prior to the approval of the R&D Strategy in 2011, funding
was divided between ‘core’ funding and project grants. Core
funding was awarded to PIs on a five year basis, subject to
satisfactory external review. Project grants of £250-500k
over 3-5 years were open to any employee. In 2011, there
were more than 25 individual project grants running, and
since most applicants were from within PI groups, the
strategy recommended that a greater proportion of funding
be allocated to PIs against strategic ‘work packages’. This
approach will produce a more focussed programme and
reduce the administrative complexity.
NHSBT will continue to have open calls for projects relating to
organ procurement and clinical studies to which non-NHSBT
staff can apply.
The allocation of funding is undertaken by the R&D
Committee of the Board, which meets twice each year and
which includes operational Directors and International experts
who provide an external view. I am very grateful to Harvey
Klein (Professor of Transfusion Medicine, National Institute
of Health, Bethesda, USA), Lucio Luzzatto (Professor of
Haematology, University of Firenze, Florence, Italy) and Rutger
Ploeg (Professor of Transplant Surgery, University Medical
Centre, Groningen, Netherlands), who have acted as our
external experts over the past three years and who give up a
good deal of time to prepare for and attend meetings. Their
input is invaluable.
Research is conducted within the DH Research Governance
Framework for Health and Social Care. NHSBT has an external
contract for Intellectual Property support, which enables
Freedom to Operate searches to be conducted as well as
patenting and exploitation of new IP.
Management of the programme has been strengthened by
creation of an Assistant Director, Research and Development
post (Dr Nick Watkins) and an enlarged R&D Senior
Management Team chaired by myself. The R&D office team
provide support and guidance to PIs. Contracts and IP have
been overseen by Professor Marion Scott in her part-time role
as R&D Manager. She will be relinquishing this role in Autumn
2012 to focus on research, and I am grateful to her and the
rest of the team for all their support during this period of
change.
Introduction
The Components Development Laboratory at Brentwood
(Head: Dr Rebecca Cardigan) and the Tissue Development
team at Liverpool (Head: Dr Paul Rooney) undertake
programmes of work on product quality and safety in close
collaboration with operational colleagues and customers.
Outputs and benchmarking
Research success in academia is conventionally measured by
external grants awarded, and the publication of high impact
papers. The embedding of PIs in Academic Institutions means
that their outputs are assessed through the National Research
Assessment Exercise. For a Blood Service, impacts on service
activities and clinical practice are also key measures, although
the translation of research findings into routine practice can
take up to a decade eg cord stem cell banking. A new R&D
group formed by the Alliance of Blood Operators will provide
an opportunity to benchmark our activity against other blood
services. Figure 3 shows annual peer-reviewed publications for
the period 2009-11 .
Publications
by Calendar
Figure 3.Peer-Reviewed
P eer-Reviewed
publications
byYear
calendar year
180
160
140
Number of publications
Development
120
100
80
60
40
20
0
2009
2010
2011
Year
Future plans
The next three-year cycle will mainly be one of consolidation
and delivery of the strategic programmes described above.
This period will also include a consultation exercise from
which the next cycle of programmes will be developed for the
NIHR funding renewal date of 2015.
The work presented in these pages confirms NHSBT’s position
at the forefront of research and development in its field.
Dr Lorna Williamson
Medical and Research Director
Research and Development Triennial Report 2009-12
9
Theme
1 Donor health and behaviour
INTERVAL study
Professor John Danesh and Professor David Roberts
CONTRIBUTING TEAM MEMBERS: INTERVAL Study Group
Professor John Danesh
Professor David Roberts
http://www.intervalstudy.org.uk/about-the-study/whos-involved
The INTERVAL study is a randomised controlled trial (RCT)
in up to 50,000 whole-blood donors recruited at the
25 donation centres across England. Over a period of
two years, participants will be randomised to give blood
either at their usual donation intervals or more frequently.
Current practice is to invite men and women to give
whole blood every 12 and 16 weeks, respectively. During
INTERVAL men will be randomised to donate every 12, 10
or 8 weeks and women every 16, 14 or 12 weeks. At the
end of the study, we will compare the amount of blood
donated and assessments of well-being between the
different study groups.
INTERVAL is important because although demand for blood
transfusions is increasing in England due to the needs of
an ageing population, blood supply is critically limited by
difficulties in attracting and retaining donors. Blood donation
services in different countries have developed varying
customs. England has a relatively long interval compared to
other European countries where the frequency is as low as
every eight weeks for men and ten weeks for women.
The study’s main objectives are to embed a RCT within the
existing framework of NHSBT that will help to shape national
donation policy by determining:
1. the optimum interval between donations, for men and
women, that maximizes blood supply without unacceptably
increasing iron deficiency/anaemia and its potential
complications
2. whether blood donation intervals can be tailored to donors
on the basis of demographic, haematological, genetic and
lifestyle factors.
During the course of the study, additional blood samples will
be taken for a full blood count and storage of plasma, serum
and DNA. These will be used to measure biomarkers as well
as genetic factors. Online questionnaires regarding health,
lifestyle and cognitive function will also be collected. A subset
of participants will take part in a study of the impact of
donation interval on physical activity levels.
10
Research and Development Triennial Report 2009-12
The Universities of Cambridge and Oxford in collaboration
with NHSBT have set up INTERVAL. The study has ethical
approval from the Cambridge (East) Research Ethics
Committee. Recruitment commenced on 11 June 2012 and
has involved the staggered roll out of donation centres at
a rate of one per week. Recruitment is scheduled to run
until March 2013 with completion of the study by April
2015. The trial will not only provide evidence for optimum
donation intervals but will also be the foundation for a future
programme of research in blood donation.
Contribution to patient and donor care or service
development
The INTERVAL study is aligned with the NHSBT’s long-term
aims to:
1. ensure the supply of blood by allowing a direct assessment
of the effect of incremental increases in donation frequency
on blood collection yields
2. improve the health and well-being of donors. Data from
this study will inform tailoring of donation intervals to
reduce deferral rates and retain donors
3. provide a more personalised approach to donation and
reduce deferral rates due to iron deficiency and anaemia,
and
4. evolve into a leading evidence-based health organisation.
Group webpage
http://www.intervalstudy.org.uk
Theme
Donor health and behaviour
1
Cambridge BioResource and National
Institute for Health Research (NIHR)
BioResource for Rare Diseases
Dr Nicola Froad
Dr Sarah Morley
Dr Jennifer Sambrook
Dr Nicola Foad, Dr Sarah Morley and Dr Jennifer Sambrook
[email protected], [email protected] and
[email protected]
CONTRIBUTING TEAM MEMBERS: Dalila Belkhiri, Sophia Coe, Abigail Crisp-Hihn, Jennifer Jolley, Heather Lloyd-Jones, Carmel Moore,
Marie Mulligan and Jonathan Stephens
Cambridge University Scientists in NHSBT have collaborated with Prof John Todd
(Fellow of the Royal Society) and Dr John Bradley of the Cambridge BioResource
and with Prof John Danesh to create the Cambridge Biomedical Research Centre
(BRC) to establish the Cambridge BioResource and Cambridge CardioResource.
About 12,000 volunteers, the majority being blood donors, have donated DNA,
plasma and serum and completed a lifestyle and health questionnaire during
Stage 1 enrolment in the Cambridge BioResource. This allows the selection and
recruitment of volunteers with a particular genotype/phenotype to Stage 2 research
studies. Researchers of the BRC and NHSBT have recalled thousands of volunteers
for Stage 2 studies resulting in 21 original publications about the association
between genes and diseases.
This success has prompted the formation of the NIHR BioResource across the BRCs of
London, Oxford and Cambridge and the Leicester Cardiovascular Biomedical Research
Unit (BRU). In addition to healthy volunteers, patients with common and rare diseases
are being enrolled in the NIHR BioResource. There are about 7,000 inherited rare
diseases, which combined affect 7% of the UK population and the causative genes of
about half have been identified. Exome sequencing and other genomics technologies
are used to identify the genes underlying the remaining diseases. Prof Ouwehand’s
group leads two studies on rare diseases, one on the formation of alloantibodies against
Rh groups during pregnancy and another on patients with unexplained bleeding and
platelet disorders.
The purpose of the first study, which is supervised by Dr Sarah Morley, is to identify
common sequence variants conferring risk of Rh alloimmunisation by a Genome-Wide
Association Scan (GWAS). This is a joint effort with Prof Ellen van der Schoot of the
Sanquin Research laboratories in Amsterdam, the Netherlands. Enrolment of the first
2,000 Rh immunised cases will be completed by the end of 2012 and the GWAS will be
performed in 2013.
RECENT PUBLICATIONS
Dendrou CA, Plagnol V, Fung E,
et al. (2009). Cell-specific protein
phenotypes for the autoimmune
locus IL2RA using a genotypeselectable human bioresource.
Nat Genet, 41, 1011-1015
Heinig M, Petretto E, Wallace C, et al.
(2010). A trans-acting locus regulates
an anti-viral expression network and
type 1 diabetes risk. Nature, 467,
460-464
Schimpl M, Moore C, Lederer C,
et al. (2011). Association between
walking speed and age in healthy,
free-living individuals using mobile
accelerometry – a cross sectional
study. Plos One, 6(8), e23299e23299
BioResource webpages
www.cambridgebioresource.org.uk
www.bridgestudy.org.uk
www.thrombogenomics.org.uk
The second study is based on collaborations with comprehensive care centres for patients with inherited bleeding and platelet
disorders at the Cambridge and London BRCs (Dr Keith Gomez, Royal Free Hospital, Prof Laffan/Dr Millar, Hammersmith Hospital,
Prof John Pasi, Barts Health NHS Hospital) and Dr Andrew Mumford from the Bristol BRU for Cardiovascular Diseases.
Overseas centres in Belgium, France, Germany, the Netherlands and the USA have joined the BRIDGE (Biomedical Research centre/
unit Inherited Diseases Genetic Evaluation Study). The coding fraction or exome of 1,000 patients will be collected and the coding
fraction or exome will be sequenced to identify the rare variants causative of the patients’ unexplained bleeding and/or platelet
disorders.
Contribution to patient and donor care or service development
The identification of genes controlling the formation of Rh alloantibodies would be a first breakthrough step in understanding why
a relatively small number of women form Rh antibodies. The BRIDGE gene discovery project is linked with the ThromboGenomics
(www.thrombogenomics.org.uk) initiative which has as its immediate aim to develop an affordable next generation based test
for the diagnosis of rare inherited bleeding and platelet disorders other than Haemophilia and von Willebrand Factor Disease.
Research and Development Triennial Report 2009-12
11
Theme
1 2
Transfusion/transplantation virology and microbiology
Transfusion microbiology with respect to the
microbiological safety of blood, tissues and organs
Professor Richard Tedder
[email protected]
CONTRIBUTING TEAM MEMBERS: Kate Tettmar, Samreen Ijaz, Philip Tuke, Poorvi Patel, Gillian Rosenberg,
Steve Dicks, Jeremy Garson, Ruth Parry, John Poh and Renata Szypulska
The projects undertaken by Professor Tedder’s group encompass elements of work
associated with the response to current and emerging microbiological threats
to the safety of blood, tissues and organs. This work is therefore responsive and
always evolving. Professor Tedder’s group sits within the joint NHS Blood and
Transplant (NHSBT) and Health Protection Agency (HPA) Blood Borne Virus Unit
based at Microbiology Services Colindale at the HPA. This joint unit provides a
unique opportunity for the sharing of expertise, data and cross training of staff.
The unit works closely with experts in both organisations who feed in to on-going
projects and collaborations.
The work undertaken by the group falls principally but not exclusively within blood
borne infections and can be divided into surveillance studies, development and
research. Current joint NHSBT/HPA and NHSBT projects include:
1. establishing the phylogeny and molecular characterisation of hepatitis viruses in
acute and chronically infected donors (B, C and E)
2. seroprevalence and molecular based studies undertaken in blood donor samples
and plasma minipools to assess the risk of acute indigenous hepatitis E infections in
donors to blood safety
3. development of ex vivo and in vitro methods for determining the biological function
of normal and mutated hepatitis B surface antigen (HBsAg)
4. a study of inducible murine retroviruses, and
5. development of an extraction method and real-time TaqMan polymerase chain
reaction assay for the detection of bacterial contamination in platelets.
An essential arm of this programme of work is the development and implementation
of methods for the detection of antibody and nucleic acid linked to current and
emerging pathogens which may impact on blood safety. A large focus currently is
the investigation of risk and outcome of transmission of hepatitis E virus (HEV) from
infected donors. This focus will examine the extent of HEV related post-transfusion
hepatitis. The outcome of receiving HEV containing components will be investigated
by following up the recipients. This study will generate data from which a risk
assessment and interventions can be implemented to limit HEV related disease by blood
transfusion.
RECENT PUBLICATIONS
Ijaz S, Szypulska R, Tettmar KI,
Kitchen A, Tedder RS, (2012).
‘Detection of hepatitis E virus RNA
in plasma mini-pools from blood
donors in England.’ Vox Sanguinis,
102:3, 272
Patel P, Garson JA, Tettmar KI,
Ancliff S, McDonald C, Pitt T,
Coelho J, Tedder RS, (2011).
‘Development of an ethidium
monoazide-enhanced internally
controlled universal 16S rDNA
real-time polymerase chain
reaction assay for detection of
bacterial contamination in platelet
concentrates.’ Transfusion, (Epub)
ahead of print
Robinson MJ, Tuke PW, Erlwein O,
Tettmar KI, Kaye S, Naresh KN,
Patel A, Walker MM, Kimura T,
Gopalakrishnan G, Tedder RS,
McClure MO, (2011). No Evidence of
XMRV or MuLV Sequences in Prostate
Cancer, Diffuse Large B-Cell
Beale MA, Tettmar K, Szypulska R,
Tedder RS, Ijaz S, (2011). ‘Is there
evidence of recent hepatitis E virus
infection in English and North Welsh
blood donors?’ Vox Sanguinis,
100:3, 340-2 Large B-Cell
Lymphoma, or the UK Blood Donor
Population. Adv. Virol.2011;782353
Contribution to patient and donor care or service development
The development of a rapid and highly sensitive molecular assay to detect bacterial contamination in platelet
concentrates.
Methods have been developed to study the genetic variation in the hepatitis B surface antigen, which can be used to
examine the clinical and functional impact of these variations including the impact on the accuracy of donor screening
assays.
A better understanding has been gained of infected blood donors through the molecular characterisation of viruses
circulating in the donor population. The data from this work informs on viral diversity as well as examining the strength
of the Donor Health Check (DHC).
Measuring the incidence of hepatitis E in blood donors and the extent of HEV related post-transfusion hepatitis
in recipients. Data from this study will contribute to appropriate risk assessments and interventions that can
be implemented to limit HEV related disease in the UK.
12
Research and Development Triennial Report 2009-12
Transfusion/transplantation virology and microbiology
Theme
1
2
Virological blood safety
Professor Jean-Pierre Allain
[email protected]
CONTRIBUTING TEAM MEMBERS: Daniel Candotti, Birgit Meldal, Penelope Garmiri, Laura Cox, Graham Freimanis,
Subhajit Biswas, Dalila Belkhiri, Mira El Chaar, Francesca Gobbini, Florence Enjalbert and Rowena Ching
Transmission of viral infections by transfusion has been at the forefront for 25 years
following the HIV-AIDS crisis. As a result, improved assays and genomic detection
has been implemented to cover HIV and hepatitis C virus infections. Hepatitis B
infection has been relatively neglected until a form of HBV infection missed by
standard serological methods and detected only by genomic, extremely sensitive
methods was identified (occult HBV infection or OBI). This laboratory has become a
world leader in this challenging viral threat.
We have collected OBI samples from all areas of the world through a large network of
blood centres. Specific methods have been developed to characterise OBIs despite very
low levels in donor circulation. These methods were applied to over 200 OBI cases as
confirmation and material for basic research.
Results provided a frequency of the condition ranging between 1:500 and 1:400,000
blood donations depending on areas of the world. It also demonstrated a 28% rate
of infectivity in blood recipients justifying efforts to continue and expand genomic
testing. The mechanisms leading to OBI were also examined and turned out to be
many and quite diverse such as a relative inefficiency of the immune system to control
the infection, the selection of mutations escaping detection by current serological
tests, abnormal biology of the virus main protein which is either not produced or not
excreted by the liver cells constituting the target of the virus, or mutations impairing the
machinery of the virus replication.
At the same time, we also studied the classical form of hepatitis B virus for comparison to
OBI in terms of both classification into genotypes and frequency of mutations that were
considerably less than in OBIs. Such studies were conducted in Ireland, Switzerland, Poland,
Italy, Spain, Iran, Turkey, Malaysia, Thailand, Ghana, Guinea, Tunisia and South Africa.
These studies significantly contributed to demonstrate the residual threat of hepatitis
B virus to blood safety, to devise the methods to identify OBIs and to justify the
implementation of HBV genomic screening in the UK.
Contribution to patient or donor care or service development
The first objective was to provide epidemiological evidence that nucleic acid testing for
HBV was justified.
RECENT PUBLICATIONS
Allain JP, Belkhiri D, Vermeulen M,
Crookes R, Cable R, Amiri A, Reddy R,
Bird A, Candotti D. Characterization
of occult Hepatitis B virus strains
in South African blood donors.
Hepatology 2009; 90: 1868-76
El Chaar M, Candotti D, Crowthers
RA, Allain JP. Impact of Hepatitis B
virus surface protein mutations on
the diagnosis of occult Hepatitis B
virus infection. Hepatology 2010;
52:1600-10
Stramer S, Wend U, Candotti D,
Forster G, Hollinger B, Dodd R,
Allain JP, Gerlich W. Occurrence
and characterization of naturally
acquired hepatitis B infection among
vaccinated blood donors. N Engl
J Med 2011: 364:236-47
Candotti D, Lin CK, Belkhiri D,
Sakuldamrongpanich T, Biswas S,
Lin S, Teo D, Ayob Y, Allain JP. Occult
hepatitis B infection in blood donors
from South East Asia: molecular
characterisation and potential
mechanisms of occurrence.
Gut. 2012 Jan 20
Group webpage
http://www.haem.cam.ac.uk/
transmed
Our second objective was to provide the means to confirm the results of blood screening and determine the criteria to classify the
type of HBV infection so that appropriate donor information can be given.
Thirdly, discoveries made at the basic science level while characterising OBIs further advanced knowledge of HBV and impacted
basic clinical issues such as quantification of HBV viral load in patients and qualification of the international standards.
Main pathways of HBV replication and particle
formation in hepatocytes. cccDNA serves as template
for transcription of both pgRNA and SmRNA. Defects in
transcription, reverse transcription, translation or export
of HBV genome or proteins can result in reduced genome
or structural proteins and be the cause of OBI. Splicing
mechanisms lead to the translation of recombinant
or neo‑protein susceptible to interfere with any of these
mechanisms.
Research and Development Triennial Report 2009-12
13
Theme
1 2
Transfusion/transplantation virology and microbiology
Reactivation of common viruses in patients receiving
chemotherapy or immunosuppressive drugs
Dr Daniel Candotti
[email protected]
CONTRIBUTING TEAM MEMBERS: Jean-Pierre Allain, Penelope Garmiri, Birgit Meldal, Francesca Gobbini and
Rowena Ching
In England, approximately 50% of blood components are transfused to patients
receiving chemotherapy for cancer or immunosuppressive drugs for bone marrow
or organ transplantation. When such patients develop a viral infection, transfusion
tends to be blamed although reactivation of a patient’s previous infections
is common and both potential origins need to be distinguished. This project
intended to determine the frequency of patient reactivation of eight common
viruses (Parvovirus B 19, Cytomegalovirus, Epstein-Barr virus, Herpesvirus-8,
varicella-zoster virus, Hepatitis B virus, Hepatitis E virus and Hepatitis A virus) when
receiving drugs such as corticosteroids, methotrexate, fludarabine, ciclosporin,
rituximab, tacrolimus or sirolimus.
The collaborative study included patients from Cambridge Dept of Haematology and
Medicine, Warsaw Institute of Haematology and Transfusion Medicine (Poland) and
the Dept. of Haematology and Transfusion in Lecco, Italy. Prior recovered infections
were identified by the presence of virus-specific antibodies and reactivation was
defined as the confirmed presence of viral genome in initially negative patients. Most
patients received preventive anti-viral treatment such as gancyclovir for herpesviruses
and lamivudine for HBV.
A total of 157 patients were studied (29 BMT, 65 liver transplantation and 63 cancer).
75-90% of patients had been exposed to four viruses (CMV, EBV, B19V, VZV),
20-40% for HAV and HEV and less than 5% for HHV-8 and HBV. Despite anti-viral
prophylaxis, 15.3% and 7.6% of exposed patients were found DNA positive for EBV
and CMV, respectively after receiving immunosuppressive drugs for variable periods
of time. None of the other viruses showed clear evidence of reactivation. Evidence of
nosocomial or community infections was found as well as effect of IV IgG received by
liver transplant patients.
Except for EBV, reactivation is relatively rare in patients receiving cancer chemotherapy
or immunosuppressive drugs. Collecting a pre-transfusion sample in these patients
would be critical to distinguish transfusion-related transmission from reactivation. This
can only be done with certainty by obtaining identical informative sequences between
blood donor and patient post-transfusion.
Contribution to patient and donor care or service development
Transfusion-transmission of viruses is a source of legal proceedings for NHSBT. This
project led to the development of qualitative and quantitative genomic assays for
eight viruses susceptible to both reactivation and transfusion-transmission. These
methods can be applied to organ, bone marrow or cord blood screening and to
the investigation of alleged transfusion-transmission of viruses in patients receiving
immunosuppressive drugs.
14
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Compston LI, Li C, Sarkodie F,
Owusu-Ofori S, Opare-Sem O,
Allain JP. Prevalence of persistent and
latent viruses in untreated patients
infected with HIV-1 from Ghana,
West Africa. J Med Virol 2009; 81:
1860-8
Power JP, El Chaar M, Temple J,
Thomas M, Spillane D, Candotti D,
Allain JP. HBV reactivation after
fludarabine chemotherapy identified
on investigation of suspected
transfusion-transmitted Hepatitis B
virus. J Hepatol 2010; 53: 780-7
Gobbini F, Owusu-Ofori S,
Marcelin AG, Candotti D, Allain JP.
Human herpesvirus 8 transfusion
transmission in Ghana, an endemic
region of West Africa. Transfusion
2012 Mar 15
Meldal BMH, Sarkodie F, OwusuOfori S, Allain JP. Hepatitis E virus
infection in Ghanaian blood donors
– the importance of immunoassay
selection and confirmation. Vox
Sanguinis 2012, in press
Group webpage
http://www.haem.cam.ac.uk/
transmed
Transfusion/transplantation virology and microbiology
Theme
1
2
Blood donors, viral infections and malaria in Ghana
Professor Jean-Pierre Allain
[email protected]
CONTRIBUTING TEAM MEMBERS: Lara Compston, Mira ElChaar, Graham Freimanis and Francesca Gobbini
The Cambridge group has conducted extensive collaboration with the Transfusion
Medicine Unit (TMU) of a major teaching hospital in Kumasi, Ghana for the past
12 years. Blood donor recruitment and blood safety were the main themes of
research. Viral safety and, in the past two years transfusion malaria, were addressed
by identifying the threats and developing strategies to reduce such threats at an
affordable cost.
Following the implementation of pre-donation serological screening for HIV, HCV
and HBV, nucleic acid testing in pools of ten donor plasma samples at an extra cost
of $4/unit was implemented. A comparison of viral infections in first-time volunteer
non-remunerated and family donors concluded the absence of difference between the
two types of donors, justifying the use of both to meet the blood demand. Partnership
between TMU and local radio stations provided 25% of the blood supply and 65%
repeat donors compared to 20% in secondary schools.
Transfusion-transmitted malaria is a neglected topic although assumed to be frequent
and clinically serious in young children and pregnant women. No screening is in place
in most of Sub-Saharan Africa. A preliminary study was conducted to determine
the frequency of antibodies to plasmodium and of parasitaemia in donor blood and
recipients as well as assessing the threat of transfusion-transmission with molecular
methods. 100% of adult donors and patients carried antibodies to plasmodium. 55%
of both donors and recipients were parasitaemic when tested with a sensitive PCR and
all four main plasmodium species were circulating although plasmodium falciparum (Pf)
was present in 90% of infections and multispecies infections were more than 10% of
total. 18-25% of non-parasitaemic recipients of parasitaemic blood were infected by
transfusion.
The failure of blood screening due to insufficient sensitivity of inexpensive tests and
the increased apparent prevalence when PCR-based tests are used impacted severely
on the blood supply. This initiated us to examine pathogen inactivation as a suitable
strategy to address transfusion malaria. The Mirasol technology applied to whole
blood units spiked with Pf monitored with a novel amplification inhibition assay and
blood culture was found efficacious with both approaches. It represents a promising
alternative to malaria testing, blood filtration, further viral and bacterial testing.
RECENT PUBLICATIONS
Owusu-Ofori S, Asenso-Mensah K,
Boateng P, Sarkodie F, Allain JP.
Fostering repeat donations in Ghana.
Biologicals 2010; 38: 47-52
Allain JP. Moving on from voluntary
non-remunerated donors: who is the
best donor? Brit J Haematol 2011;
154, 667–785
Freimanis G, Sedegah M,
Owusu-Ofori S, Kumar S, Allain JP.
Investigating the Prevalence of
Transfusion-transmission of
Plasmodium within a hyperendemic
blood donation system. Transfusion
accepted for publication
Ala F, Allain JP, Bates I, Boukef K,
Boulton F, Brandful J, Dax EM, ElEkiaby
M, Farrugia A, Gorlin J, Hassall O,
Lee HH, Loua A, Maitland K,
Mbanya D, Mukhtar Z, Murphy W,
Opare-Sem O, Owusu-Ofori S,
Reesink H, Roberts D, Torres O,
Totoe G, Ullum H, Wendel S.
External financial aid to blood
transfusion services in sub-Saharan
Africa – A need for reflection.
PLOs Medicine, in press
Group webpage
http://www.haem.cam.ac.uk/
transmed
Contribution to patient and donor care or service development
This project does not bring direct contribution to NHSBT although through
the Blood and Organ Transmissible Infectious Agents (BOTIA) donor-recipient
repository a critical tool to study emerging infections possibly extending to the UK
is of major interest to the blood service.
The amplification inhibition assay developed for Pf can be applied to any other
pathogen targeted by a pathogen inactivation technique.
Log inhibition of P falciparum genome amplification following treatment with the
Mirasol system.
A. Impact of Riboflavin and UV treatment on plasmodium genomic amplification
inhibition at two different exposure levels: 80 and 160J/ml RBC.
B. Impact of UV exposure on plasmodium genomic DNA at increasing level of UV
exposure: 40, 80, 120 and 160J/ml RBC.
Research and Development Triennial Report 2009-12
15
Theme
1 2
Transfusion/transplantation virology and microbiology
A screening test for anti-malarial antibodies
Professor David Roberts
[email protected]
CONTRIBUTING TEAM MEMBERS: David J Smith, Rozieyati Mohammed-Saleh, Adam Richie and
John Burthem (Univ Manchester)
Although rare, transfusion-transmitted malaria is frequently lethal if not treated immediately. Almost all such cases of malaria
originate from malaria-infected donors returning from malaria-endemic regions. There is a clear need for NHSBT and other
blood services to identify and exclude asymptomatic malaria infected donors while minimising the exclusion of uninfected
donors. We are aiming to develop a test that reliably detects infection with all four species of human malaria.
All donors travelling to tropical areas where malaria is present cannot donate
immediately on their return but can restart giving blood after a six-month
deferral period and after a negative specific malaria antibody test. At present,
several different malaria antibody diagnostic tests are commercially available but
they all have their limitations.
Many of these tests detect malarial antibodies against plasmodial proteins that
are potential malaria vaccine candidates and others have inadequate sensitivity
to detect infection with other human species of malaria, such as P. ovale and
P. malariae. As such there is scope to develop an alternative test to replace the
current tests should they become obsolete if the vaccine candidates become
widely used, to improve the sensitivity and specificity of testing and for
confirmation of positive results.
At the moment the United States Food and Drug Administration (FDA) has not
approved current tests for clinical use but has repeatedly said that only tests
with antigens from all four major species of human malaria would be acceptable
for FDA approval.
We have used an unbiased screening proteomic approach to identify target
P. falciparum proteins that are recognised by cohorts of malaria immune sera
but that do not react with non-immune sera. The identified blood stage target
antigens have been cloned and expressed as recombinant proteins in a suitable
bacterial system.
To date, 15 target proteins have been expressed with 14 of them successfully
purified. An ELISA test using the proteins to capture and detect specific malarial
antibodies from human serum has been developed and used to assess the
ability of the purified proteins to distinguish non-infected and malaria infected
serum samples. Proteins have been tested singly or in combination to determine
those with the most effective diagnostic value. The best three antigens give
similar if not better results to those in commercial tests and this strategy can
now be used to pick out similar antigens from other malaria species that can be
incorporated into a test to detect blood donors carrying malaria.
Figure 1. Target antigens for a malaria test
can be identified by immune-precipitation
and isolation of individual proteins on a 2-D
gel. Individual proteins are digested with
trypsin and identified after mass-spectrometry
by a database search for the charge/mass
ratio (using MALDI/TOF) and sequencing of
individual fragments (using tandem MS).
Contribution to patient and donor care or service development
Screening for active malaria infections in donors who have returned from malaria-endemic countries is vital for maintaining the
donor pool and eliminating the risk of transfusion-transmitted malaria infections in blood recipients. The current testing strategy is
an effective means of ensuring the safety of the blood supply, however, it does have some limitations. We have a new strategy to
define malarial antigens from different malaria parasites for a test that will reliably detect infection with all four species of human
malaria for use worldwide.
Group webpage
http://www.ndcls.ox.ac.uk/ResGroups.php?GID=3
16
Research and Development Triennial Report 2009-12
Transfusion/transplantation virology and microbiology
Theme
1
2
Non-coding RNAs in cytomegalovirus infection
Dr Lars Dölken
[email protected]
CONTRIBUTING TEAM MEMBERS: Anne L’Hernault, Andrzej Rutkowski and Miranda de Graaf
Humans are infected with eight different herpesviruses responsible for a broad
range of disease ranging from the common cold sore to cancer. All of us are
infected with at least three of these viruses. Upon primary infection, they all
establish a life-long, latent infection posing the constant risk of reactivation and
disease. Herpesviruses inflict a substantial burden of disease and economic costs in
transplantation and transfusion medicine, in immunocompromised patients as well
as in pregnancy.
Recently, herpesviruses were found to express small non-protein-coding RNA molecules
termed microRNAs. Our work aims at characterising the function and suitability of
these molecules for targeting by urgently needed novel antiviral agents.
MicroRNAs (miRNAs) are small RNA molecules, which regulate protein expression by
sequence-specific binding to target RNA molecules. To date, more than 1,000 miRNAs
have been identified in humans. Recent pharmacological developments now allow
them to be readily targeted using anti-sense molecules, so called ‘AntagomiRs‘. As
such, an AntagomiR against cellular miR-122, a cellular miRNA of crucial importance
for hepatitis C virus (HCV) replication, has successfully entered clinical trials for HCV
treatment. Herpesvirus encoded miRNAs (See Table 1) thus represent interesting
candidates for novel antiviral agents.
Atlas of viral miRNA targets
Our research is focussed on providing a comprehensive understanding of their complex
functions. Recently, we published the first atlas of viral miRNA targets for two of the
eight human herpesviruses, namely Kaposi’s sarcoma-associated herpesvirus and
Epstein-Barr-Virus, the causative agent of glandular fever. On the one hand, these
miRNAs aid the evasion of our immune system, promote the development of tumours
and prevent cell death. On the other hand, they enhance the ability of these viruses to
establish a persistent infection, remain quiescent for years and prevent uncontrolled
virus reactivations (see Figure 1).
As many viral miRNA targets are involved in innate and adaptive immunity, animal
models are required to study their function. Therefore, we established the murine
cytomegalovirus (mCMV) miRNA model. We identified two mCMV miRNAs to
be important for long-term persistent infection and host-to-host spread – the first
phenotype of a viral miRNA knock-out virus in vivo. In addition, we found efficient
mCMV replication to be dependent on the elimination of two cellular miRNAs by a
novel viral large non-coding RNA. The underlying molecular mechanism remains to be
elucidated.
In summary, by combining systems biology technologies with animal models this work
aids to provide the knowledge required to design novel antiviral strategies to prevent
herpesvirus reactivation or target the latent virus reservoir.
Contribution to patient and donor care or service development
RECENT PUBLICATIONS
Degradation of cellular mir-27 by a
novel, highly abundant viral transcript
is important for efficient virus
replication in vivo. Marcinowski L,
Tanguy M, Krmpotic A, Rädle B,
Lisnić VJ, Tuddenham L,
Chane-Woon-Ming B, Ruzsics I,
Erhard F, Benkartek C, Babic M,
Zimmer R, Trgovcich J, Koszinowski
UH, Jonjic S, Pfeffer S, Dölken L.
PLoS Pathogens 2012
Feb;8(2):e1002510
Small RNA deep sequencing
identifies microRNAs and other
small noncoding RNAs from human
herpesvirus 6B. Tuddenham L,
Jung JS, Chane-Woon-Ming B,
Dölken L, Pfeffer S. Journal of
Virology 2012 Feb;86(3):1638-49
Cytomegalovirus microRNAs facilitate
persistent virus infection in salivary
glands. Dölken L, Krmpotic A,
Kothe S, Tuddenham L, Tanguy M,
Marcinowski L, Ruzsics Z, Elefant N,
Altuvia Y, Margalit H,
Koszinowski UH, Jonjic S, Pfeffer S.
PLoS Pathogens 2010 Oct
14;6(10):e1001150
Dölken L, Malterer G, Erhard F, Kothe
S, Friedel CC, Suffert G, Marcinowski
L, Motsch N, Barth S, Beitzinger M,
Lieber D, Bailer SM, Hoffmann R,
Ruzsics Z, Kremmer E, Pfeffer S,
Zimmer R, Koszinowski UH, Grässer F,
Meister G, Haas J. Systematic analysis
of viral and cellular microRNA targets
in cells latently infected with human
gamma-herpesviruses by RISC
immunoprecipitation assay. Cell Host
Microbe. 2010 Apr 22;7(4):324-34
Group webpage
http://www.med.cam.ac.uk/dolken
This work comprises basic research on the functional role of herpesvirus non-coding
RNA and their role in virus persistence and reactivation. A better understanding of the molecular mechanisms involved in these
fundamental processes pathognomic for all members of the herpesvirus family may enable us to develop novel therapies to
reduce the viral load in transplant patients prior to reactivation or prevent the occurrence thereof. This would substantially reduce
herpesvirus-related morbidity and graft-failure in transplant patients as well as the related costs for pre-emptive monitoring and
treatment.
Triennial Research and Development Report 2009-12
17
Theme
1 2
Transfusion/transplantation virology and microbiology
Figure 1.
Table 1
18
Virus
Number of miRNAs
Herpes simplexvirus 1
16
Herpes simplexvirus 2
17
Varizellazoster virus
0
Epstein-Barr-Virus
25
Human cytomagalovirus
12
Human herpesvirus 6
3
Human herpesvirus 7
not studied
Kapasi’s sarcoma hepesvirus
12
Research and Development Triennial Report 2009-12
Transfusion/transplantation virology and microbiology
Theme
1
2
Transfusion medicine epidemiology review: TMER
Dr Patricia Hewitt
[email protected]
CONTRIBUTING TEAM MEMBERS: Charlotte Llewelyn, Jan Mackenzie (NCJDSRU) and Robert Will (NCJDSRU)
The Transfusion Medicine Epidemiology Review (TMER) uses the National CJD
Surveillance (NCJDSU) Register and UK Blood Service records to see whether:
a) patients with Creutzfeldt-Jakob Disease (CJD) may have given blood donations
and, if so, whether any patient receiving that blood is also known to have CJD,
and
b) whether any patient with CJD has ever received a blood transfusion and, if so,
whether any of the donors are known to have CJD.
As of 31 March 2012, 18/176 variant CJD cases gave blood donations from which
blood components were issued to hospitals. Of 67 recipients identified, 18 are currently
alive and 49 dead, including four cases of probable transfusion-transmitted infection
(three clinical cases and one pre- or sub clinical case) associated with transfusion of
non-leucodepleted red cells (1996-1999) originating from three different donors. All
live recipients have been informed of their exposure and asked to take precautions to
prevent passing on vCJD to other people eg not donate blood, tissues or organs.
A retrospective case-note review of deceased recipients of vCJD implicated
components found no evidence that any individuals (other than the three clinical vCJD
cases) had expressed clinical signs or symptoms suggestive of vCJD during life, but only
four recipients had survived more than five years post transfusion.
10/176 vCJD cases had transfusion records, including the three known cases. One was
transfused at the onset of symptoms. None of the 112 donors traced who had given
blood to the remaining six cases have developed vCJD. A report detailing a possible link
to a common donor between one case transfused at birth and another vCJD case was
published, but this was probably due to chance.
RECENT PUBLICATIONS
Gilles M, Chohan G, Llewelyn AC,
Mackenzie J, Ward HJT, Hewitt PE,
Will RG. A Case-note review
of deceased recipients of vCJD
implicated blood. Vox Sanguinis
2009; 97:211-218
Ward HJT, Mackenzie JM,
Llewelyn CA, Knight RSG, Hewitt PE,
Connor N, Molesworth A, Will RG,
Variant Creutzfeld-Jakob disease and
exposure to fractionated products.
Vox Sanguinis 2009; 97:207-210
Chohan G, Llewelyn C, Mackenzie J,
Cousens S, Kennedy A, Will W,
Hewitt P. Variant Creutzfeld-Jakob
disease in a transfusion recipient:
coincidence or cause? Transfusion
2010; 50:1003-1006
Group webpage
http://www.cjd.ed.ac.uk/TMER/
TMER.htm
Twenty-five units of plasma donated before 1998 by donors who later developed vCJD were sent for UK fractionation into 191
plasma product batches and a separate exercise by the Health Protection Agency was undertaken to trace recipients. To date, none
have developed vCJD. However, abnormal prion protein was detected at post-mortem in the spleen of one haemophilia patient
given Factor VIII, but who died of causes unrelated to vCJD.
A case-note review in 2009 showed that 9/168 vCJD cases on the CJD Surveillance Unit (SU) Register had received low risk
fractionated plasma products on a total of 12 occasions. It was unlikely that any of these cases were infected through this route,
but the possibility that such transmission could occur in future cannot be ruled out.
No links have emerged so far to suggest any evidence of sporadic CJD or familial CJD transmission by blood transfusion, but this
important work is on-going.
Contribution to patient and donor care or service development
Although the vCJD epidemic has peaked, evidence suggests there is
a population of asymptomatic ‘carriers’ of vCJD who may or may not
develop symptoms. In the absence of a validated screening test to identify
infectivity in blood, this sub-clinical population poses a major public health
concern. The TMER has fed into a series of UK measures to minimise
the risk of vCJD transmission by blood transfusion; the latest being a
recommendation in 2009 by the Advisory Committee on the Safety of
Blood Tissues and Organs (SaBTO) on the introduction of prion-filtered
blood cells for some or all groups of patients. A final decision from the
Department of Health is awaited.
Research and Development Triennial Report 2009-12
19
Theme
1 2
Transfusion/transplantation virology and microbiology
Screening assays for Prion disease
Professor David Anstee
[email protected]
CONTRIBUTING TEAM MEMBERS: Gary Mallinson, Nigel Appleford and Maria Kaisar
The human prion disease variant Creutzfeldt-Jakob disease (vCJD) is an incurable,
fatal disease of the brain. It can be transmitted to blood, organ and tissue recipients
from donors who show no signs or symptoms of the disease and, because the
incubation period for vCJD is very long, donors may not do so for many months
or years. One donor could potentially spread the disease to many individuals and
pose a risk to public health and the public trust in blood and organ donation. The
development of blood screening tests would be an important step in protecting
recipients of blood, blood products, organs and tissues from vCJD.
A number of good, reliable tests have been developed to detect the agent thought to
cause the disease (known as a prion). We have developed a test to screen NHSBT and
Scottish National Blood Transfusion Service (SNBTS) tissue donors for vCJD and are
currently conducting a two year study evaluation trial. We have recently extended the
testing to screen for CJD in cornea donors. For these tests we are using tissue samples
where vCJD prion is likely to be present at a relatively high level before the disease
symptoms occur.
In the blood of persons who are incubating vCJD and even in persons with clinical
vCJD, prions are present at very low levels and well below the limits of detection of
conventional assays.
Recently, it has been possible to detect prions in the blood of experimentally prion
innoculated animals by a technique that amplifies the prion in blood. The technique,
known as Quaking-Induced Conversion of prion (QuIC) can detect prion from vCJD
brain samples at 1014 dilution in blood. We are developing QuIC to a form suitable for
use as a blood screening test and to facilitate this we are collaborating with the Roslin
Institute, University of Edinburgh to use a sheep model as close as possible to vCJD in
humans. Using this model we can determine how early in the incubation period we
can detect prions in blood and help assess the effectiveness of prion reduction measures
such as leucodepletion and prion filtration of blood.
Contribution to patient and donor care or service development
The CJD screening assay for NHSBT & SNBTS tissue donors and cornea donors has
reduced the risk of transmission of CJD to transplant recipients of tissues such as skin,
bone, heart valves and cornea.
The development of the QuIC assay will help the UK blood services assess the risk of
vCJD transmission in humans through blood transfusion and other organ and tissue
donations.
RECENT PUBLICATIONS
McGuire LI, Peden AH, Orrú CD,
Wilham JM, Appleford NE,
Mallinson G, Andrews M, Head MW,
Caughey B, Will RG, Knight RS,
Green AJ. RT‑QuIC analysis of
cerebrospinal fluid in sporadic
Creutzfeldt‑Jakob disease Annal
Neurol. 2012. doi:10.1002/ana.23589
Peden AH, McGuire LI, Appleford NE,
Mallinson G, Wilham JM, Orrú CD,
Caughey B, Ironside JW, Knight RS,
Will RG, Green AJ, Head MW. Sensitive and specific detection of
sporadic Creutzfeldt-Jakob disease
brain prion protein using real-time
quaking-induced conversion.
J Gen Virol. 2012 Feb;93(2):438-49.
Epub 2011 Oct 26
Warwick RM, Armitage WJ,
Chandrasekar A, Mallinson G,
Poniatowski S, Clarkson A. A pilot to
examine the logistical and feasibility
issues in testing deceased tissue
donors for vCJD using tonsil as the
analyte. Cell Tissue Bank. 2012
Mar;13(1):53-61. Epub 2010 Nov 3
Clewley JP, Kelly CM, Andrews N,
Vogliqi K, Mallinson G, Kaisar M,
Hilton DA, Ironside JW, Edwards P,
McCardle LM, Ritchie DL,
Dabaghian R, Ambrose HE, Gill ON.
Prevalence of disease related
prion protein in anonymous tonsil
specimens in Britain: cross sectional
opportunistic survey. BMJ. 2009
May 21;338:b1442. doi: 10.1136/
bmj.b1442
Group webpage
http://ibgrl.blood.co.uk
Quaking-Induced Conversion (QuIC) analysis of vCJD brain and
spleen homogenates diluted into human plasma.
20
Research and Development Triennial Report 2009-12
Theme
Appropriate and safe use of blood components 1
3
Transfusion alternatives before surgery in
Sickle Cell Disease (TAPS)
Dr Lorna Williamson
[email protected]
CONTRIBUTING TEAM MEMBERS: Jo Howard, Tony Johnson (Medical Research Council), Charlotte Llewelyn,
Moira Malfroy, Louise Choo (CSU team) and Sally Davies
Patients with sickle cell disease (SCD) can develop complications such as acute
painful crisis or acute chest syndrome (ACS) after planned surgery. A UK survey
we carried out in 2005 showed that there was no consensus as to whether SCD
patients should be transfused before undergoing low or medium risk surgery.
Between 2007-11, we carried out a multi-centre randomised controlled trial (TAPS)
in UK, Canada and the Netherlands, which recruited adults and children aged over
one year scheduled to have low or medium risk surgery. They were randomised
to receive no pre-operative transfusion (Arm A) or to receive either a top-up or
exchange transfusion, depending on the haemoglobin level (Arm B). We compared
the proportion of patients who in each arm who developed clinically significant postoperative complications, and an independent safety committee monitored serious
adverse events (SAEs).
RECENT PUBLICATIONS
Howard J, Malfroy M, Llewelyn C,
Choo L, Rees D, Walker I, Tilyer L,
Fijnvandraat, K, Kirby-Allen M,
Hodge R, Purohit S, Davies SC,
Williamson LM. Pre-Operative
Transfusion Reduces Serious
Adverse Events in Patients with
Sickle Cell Disease (SCD): Results
from the Transfusion Alternatives
Pre-operatively in Sickle Cell Disease
(TAPS) Randomised Multi-Centre
Clinical Trial. Blood (2011), Vol 118;
Issue 21, Abstract 9
In March 2011 the Trial Steering Committee took the decision to close the trial early
after an excess of patients experiencing SAEs was reported in Arm A (n=10) compared
to Arm B (n=1). All but one of these 11 patients had ACS, and, although all patients
recovered, the risks of this serious complication were considered unacceptable. Results
were then analysed from 67/70 patients recruited. Thirteen of 33 of patients (39%)
in Arm A developed post-operative complications compared to only 15% (5/34) of patients in Arm B, which was significantly
different (P=0.023). Twelve of the patients who were not transfused pre-op subsequently needed to have a transfusion during or
after surgery, as a result of a complication (mostly ACS), compared to only three patients in Arm B.
Only one patient (in Arm B) had developed a new alloantibody (Anti-S) following transfusion. No differences in length of hospital
stay or re-admission rates were seen between the two groups.
It was concluded that pre-operative transfusions should be offered to sickle cell patients (Hb SS) before low or medium risk surgery.
The results were presented at the American Society for Haematology in December 2011, when it was selected for both the press
briefing and ‘ASH on tour’, a post-conference video presentation. A manuscript has been submitted for publication, and a health
economic analysis will follow.
Contribution to patient and donor care or service development The trial is being taken into account in the formulation of the new British Committee for Standards in Haematology guidelines for
transfusion in haemoglobinopathy patients.
Research and Development Triennial Report 2009-12
21
Theme
1
3
Appropriate and safe use of blood components
Studies into the clinical effectiveness of platelet
transfusions and the optimal product
Dr Simon J Stanworth
[email protected]
CONTRIBUTING TEAM MEMBERS: Clinical Studies Unit team. Systematic Reviews Initiative team
RESEARCHERS: Lise Estcourt, Anne Kelly, Rebecca Cardigan, Sheila MacLennan, Cristina Navarrete, Judith Marsh,
Ana Curley and Mike Murphy
One of NHSBT’s clinical research programmes focuses on the use of platelet
transfusions. This was given high priority by NHSBT’s appropriate and safe use of
blood strategy group because of the rapid increase in hospital platelet usage and
the high level of inappropriate use identified in our recent national audit.
We have updated the Cochrane systematic review of platelet usage, to incorporate
new data from platelet dose studies. The Clinical Studies Unit (CSU) has supported
a large randomised controlled trial (TOPPS) in adult patients with blood cancers to
compare prophylactic platelet transfusion using a platelet threshold of 10x109/L with a
‘no-prophylaxis’ arm, in which patients receive platelets only if bleeding or for clinical
reasons. The primary outcome measure is the proportion of patients having a significant
clinical bleed up to 30 days from randomisation. Analysis has started and results will be
presented at international meetings over the coming year.
The value of prophylactic platelets is equally uncertain in neonates, who are another
intensively transfused group of patients. We have started a randomised clinical trial
(PLANET-2) comparing two different platelet counts as triggers for platelet transfusion.
The primary outcome is a composite of mortality and major bleeding at 28 days after
randomisation. Importantly, we have shown that consenting and randomising babies to
trials of blood transfusion is feasible.
Best products for transfusion
The CSU is also undertaking studies to
address the best product for transfusion. A
trial of extended shelf-life platelets (PPIP) has
been completed in 2012 and will provide
data to compare increases in platelet count
in patients who have received either 2-5 or
6-7 day old platelets. The PROMPT study
examines whether the characteristics of
apheresis platelets affect outcomes in 100
patients with blood cancers. It is looking
at whether platelets that are characterised as being of ‘high’ reactivity are different
to platelets characterised as being of ‘low’ reactivity in their effect on platelet count
increment. This trial is open to recruitment.
Finally, The HLA epitope study is designed to compare the effect of HLA epitopematched (HEM) with standard HLA-matched (HSM) platelet transfusions in raising the
platelet count (CI) in patients with types of blood cancers. It will tell us about different
ways of providing platelets for patients who unfortunately have antibodies to proteins
on platelets, which makes transfusion more difficult. This study is due to commence in
September 2012.
RECENT PUBLICATIONS
Estcourt L, Stanworth S, Doree C,
Hopewell S, Murphy MF, Tinmouth A,
Heddle N. Prophylactic platelet
transfusion for prevention of bleeding
in patients with haematological
disorders after chemotherapy and
stem cell transplantation. Cochrane
Database Syst Rev. 2012 May
16;5:CD004269
Estcourt LJ, Stanworth SJ, Murphy MF.
Platelet transfusions for patients with
haematological malignancies: who
needs them? Br J Haematol 2011
Aug;154 (4): 425-40
Stanworth SJ, Dyer C, Choo L,
Bakrania L, Copplestone A,
Llewelyn C, Norfolk D, Powter G,
Littlewood T, Wood EM, Murphy MF;
TOPPS Study Group. Do all patients
with hematologic malignancies and
severe thrombocytopenia need
prophylactic platelet transfusions?
Background, rationale, and design
of a clinical trial (trial of platelet
prophylaxis) to assess the effectiveness
of prophylactic platelet transfusions.
Transfus Med Rev. 2010 Jul;24(3):
163-71
Stanworth SJ, Clarke P, Watts T,
Ballard S, Choo L, Morris T,
Murphy MF, Roberts I; for the Platelets
and Neonatal Transfusion Study
Group. Prospective, Observational
Study of Outcomes in Neonates With
Severe Thrombocytopenia. Pediatrics
2009 124(5);e826-e834
Contribution to patient and donor care or service development
Clinical studies of the use of prophylactic platelet transfusions by comparison to no transfusions will provide fundamental insight
into the overall value of platelet transfusions to prevent bleeding in patients with types of blood cancer. The results may help tell
us which patients do not need to receive platelet transfusions when they are showing no signs of bleeding. There are very few
platelet transfusion studies in infants, and the study of platelet transfusions in neonates will provide evidence for safe practice in a
very vulnerable group of patients. We need to know whether platelets stored for a longer period of time are safe for patients, as
this is important for managing our stocks of platelets for hospitals.
22
Research and Development Triennial Report 2009-12
Theme
Appropriate and safe use of blood components 1
3
Use of information technology to deliver safer and
more effective transfusion practice
Professor Mike Murphy
[email protected]
CONTRIBUTING TEAM MEMBERS: Julie Staves, Edward Fraser, Simon Noel, Barbara Cripps, Jonathan Kay and
Paul Altmann
There remains considerable potential to further improve hospital transfusion practice
so there are fewer ‘wrong blood’ errors, less inappropriate use of blood and
improved efficiency of the hospital transfusion process including rapid provision of
blood for patients that need it urgently, reduced staff time, less blood wastage and
simpler compliance with national regulations for clinical and laboratory transfusion
practice.
Research and development activities
An electronic blood transfusion management system was developed in 2001 at
the Oxford University Hospitals (OUH) supported by NHSBT and fully implemented
throughout the OUH in 2006. It facilitates safer practice, saves time and reduces costs.
It was one of the first six recommended interventions for NHS Trusts as part of the NHS
Quality, Innovation Productivity and Prevention (QIPP) programme, and has won many
national awards, including the Health Service Journal Award for Improving Care with
Technology and UK IT Industry Awards for Public Sector Project of the Year in 2009.
We have recently demonstrated the benefit of the system for active monitoring of
transfusion practice and driving better practice.
There is considerable potential for other centres to take advantage of the technology
we have developed for transfusion and the process for ‘electronically controlled remote
blood issue’, which enables us to provide a ‘centralised transfusion service’ (CTS) for
Oxfordshire. The Oxford CTS provides a model for CTS elsewhere in the UK, and indeed
worldwide.
Further developments being explored include:
1. a ‘decision support’ module to provide guidance to clinicians ordering blood to
promote adherence to guidelines for the appropriate use of blood and further
reduce costs
2. a ‘data mining’ tool to provide clinical teams with regular comparative data on their
blood usage
3. the inclusion of information of intra-operative cell salvage and near-patient
haemostasis testing to increase the effectiveness of blood conservation activities,
and
RECENT PUBLICATIONS
Murphy MF, Fraser E, Miles D, Noel S,
Staves J, Cripps B, Kay J, (2012). How
do we monitor hospital transfusion
practice using an end-to-end
electronic transfusion management
system? Transfusion Jan 9. doi:
10.1111/j.1537-2995.2011.03509.x.
(Epub ahead of print)
Murphy MF, Staves J, Davies A,
Fraser E, Parker R, Cripps B, Kay J
& Vincent C, (2009). How do
we approach a major change
program using the example of
the development, evaluation, and
implementation of an electronic
transfusion management system.
Transfusion. 49, p829-837
Staves J, Davies A, Kay J, Pearson, O,
Johnson T & Murphy MF, (2008).
Electronic remote blood issue:
a combination of remote blood
issue with a system for end-to-end
electronic control of transfusion to
provide a “total solution” for a safe
and timely hospital blood transfusion
service. Transfusion. 48, p415-424
Murphy MF, Stanworth SJ, &
Yazer M, (2011). Transfusion practice
and safety: current status and
possibilities for improvement. Vox
Sang. 100, p46-59
4. linkage with other clinical IT developments in Oxford such as
active patient monitoring and medicines management.
Contribution to patient and donor care or service
development
• national and international exemplar of the use of technology to
improve clinical care (highlighted by update of our work in the
NHS QIPP programme www.evidence.nhs.uk/qipp)
• national and international model for modernisation of
transfusion services, and
• contribution to NHSBT’s Integrated Transfusion Services
programme.
A selection of the awards won by Professor Murphy and
his team (see page 62).
Research and Development Triennial Report 2009-12
23
Theme
1
3
Appropriate and safe use of blood components
Systematic Review Initiative (SRI)
Professor Mike Murphy
[email protected]
CONTRIBUTING TEAM MEMBERS: Simon Stanworth, Susan Brunskill, Dr Carolyn Dorée, Sheila Fisher,
Dave Roberts, Nikki Curry, Lise Estcourt, Vipul Jairath, Chris Hyde , Sally Hopewell and Marialena Trivella
The strategic aim of the SRI is to strengthen the evidence base of transfusion
medicine by initiating, supporting and disseminating systematic reviews of both
the randomised and non-randomised controlled trial literature. The work is linked
to the development of new clinical trials and underpinning and/or challenging
recommendations for practice.
Publication of systematic reviews
In the last three years we have published 16 systematic reviews, including systematic
reviews of granulocytes for preventing infection, donation by stem cell or bone marrow:
the donor experience, the role of the patient in blood transfusion safety, an overview
of the randomised controlled trials evidence base for red blood cell transfusion and two
reviews exploring the evidence for the bedside practice of transfusion medicine.
Seven of our systematic reviews have been in collaboration with The Cochrane
Collaboration and five have been significant updates of previously published systematic
reviews (recombinant factor VIIa, antenatal interventions for fetomaternal alloimmune
thrombocytopenia, platelet transfusions for patients with haematological malignancy,
stem cell treatment for acute myocardial infarction and the use of fresh frozen plasma).
Impact of our systematic reviews
Of our reviews, eight have contributed to the development of at least one UK or
international guideline for example our reviews of the acute management of trauma
hemorrhage, trauma induced coagulopathy and recombinant factor VIIa were all
included in the Canadian National Advisory Committee on Blood and Blood Products Massive Transfusion Consensus Conference and report of the panel 2011 and a number
of others have formed the foundation for significant laboratory and clinical studies.
Transfusion Evidence Library
Another crucial area of our work is to identify and disseminate all published randomised
controlled trials and systematic reviews in transfusion medicine. We launched our
Transfusion Evidence Library (www.transfusionevidencelibrary.com) in September
2009. This is a fully searchable online database containing references to 780 high quality
transfusion medicine systematic reviews, plus 1,520 fully indexed, randomised controlled
trials obtained through hand searching conference proceedings from 1980 onwards.
The library is updated monthly and over the coming months will contain references to
relevant randomised controlled trials identified from comprehensive searches of Medline.
In 2011 a logo was developed for the library and since then the library has permanent
online links from four medical societies and one journal website. We are seeking to
increase the dissemination of the library through 2012.
Contribution to patient and donor care or service development
• eight of our systematic reviews have been used to develop and guide practice in at
least one UK or international guideline, and
• our review of the safety of blood donation from individuals with treated
hypertension or non-insulin dependent type 2 diabetes (2010) led to a change in
practice in NHSBT and inclusion of these patients as blood donors.
A number of our reviews, for example, trauma induced coagulopathy, fresh frozen
plasma and prophylactic platelets in haematological malignancy have formed the
foundation for significant ongoing laboratory, audits and clinical studies
24
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Simpson E, Lin Y, Stanworth
S, Birchall J, Doree C, Hyde C.
Recombinant factor VIIa for the
prevention and treatment of bleeding
in patients without haemophilia.
Cochrane Database of Systematic
Reviews 2012, Issue 4: CD005011
Brunskill S, Thomas S, Whitmore E,
McDonald CP, Doree C, Hopewell
S, Staves J, Cardigan R, Murphy MF.
What is the maximum time that a
unit of red blood cells can be safely
left out of controlled temperature
storage? Transfusion Medicine
Reviews Epub 2011: Nov 25
Wilkinson KL, Brunskill SJ, Doree C,
Hopewell S, Stanworth S, Murphy
MF, Hyde C. The clinical effects of red
blood cell transfusions: an overview
of the randomised controlled trials
evidence base. Transfusion Medicine
Reviews 2011; 25 (2): 145-155, e2
Curry N, Hopewell S, Doree C,
Hyde C, Brohi K, Stanworth S.
The acute management of trauma
haemorrhage: A systematic review of
randomised controlled trials. Critical
Care 2011; 15(2) R92
Transfusion Evidence Library can
be accessed:
http://www.
transfusionevidencelibrary.com
Group webpage:
http://www.ndcls.ox.ac.uk/
ResGroups.php?GID=11
Theme
Appropriate and safe use of blood components 1
3
NHSBT/Medical Research Council – Clinical Studies Unit
Dr Charlotte Llewelyn
[email protected]
CONTRIBUTING TEAM MEMBERS: CSU Team: Alison Deary, Claire Dyer, Lise Estcourt, Fiona Goddard, Lekha Bakrania,
Renate Hodge, Gillian Powter, Moira Malfroy, Kay Harding, Ana Mora, Tania Reed, Rupa Sharma, Jayne Gipp, Sue Barton,
Louise Choo (MRC), Brennan Kahan (MRC), Anne Kelly, Catherine O’Donovan, Lynn Fraser, Diane Forrest, Beverley Jones,
Heather Smethurst, Karen Willoughby, Sobiya Nadaraja (Kings), Simon Stanworth, Mike Murphy and Lorna Williamson
RESEARCHERS: Nikki Curry, Vipul Jairath and Gemma Simons
Between 2009-12 the Clinical Studies Unit (CSU) has worked on 26 trials. Eight
trials are currently open to patient recruitment, five more are due to start in mid
2012, and 13 have been completed and are at varying stages of analysis and
write up. Seventeen papers have been published relating to these and earlier CSU
studies.
A major focus has been the completion of two multi-centre international randomised
controlled trials: Transfusion Alternatives in Preoperative sickle cell patients (TAPS) and
a study of prophylactic platelet transfusion in patients with blood cancers (TOPPS),
(see Williamson, page 21 and Stanworth, page 22). The CSU has also supported
several studies to assess new blood components:
1. the Platelet Process Improvement Project (PPIP) randomised controlled trial to assess
bleeding scores and increments comparing 2-5 day-old and bacterially screened
6-7 day old platelets
2. a cohort safety study (PRISM-A) demonstrated that transfusion of prion-filtered red
cells to surgical patients did not increase the rate of alloimmunisation or transfusion
reactions, and
3. the granulocytes in neutropenia (GIN-1) observational study showed that pooled,
whole blood-derived granulocytes in additive solution and plasma had a similar
safety profile to existing sources of granulocytes in neutropenic patients.
New trials and a pilot study
The Appropriate Use of Blood Strategy Group has agreed that new trials should
fall mainly into two main themes: trials on clinical effectiveness of platelets (see
Simon Stanworth, page 22), and trials in bleeding patients. The latter includes a
feasibility study to investigate effects of early administration of cryoprecipitate to
patients with major traumatic haemorrhage (CRYOSTAT), a pilot study to examine
the usefulness of haemostatic ‘point of care’ tests in critical care patients with
coagulopathy (ISOC-2), and a cluster randomised trial (TRIGGER) which will compare
two haemoglobin thresholds for transfusing red cells to patients with gastro-intestinal
bleeding.
The CSU will continue to contribute to international efforts led by others, including a
randomised controlled age of blood trial (ABLE) and NEPTUNIS, a survey of outcomes
of neutropenic sepsis.
During 2012-13, there will be a planned merger between the CSU and NHSBT’s
Statistics and Clinical Audit team. This will facilitate broadening of CSU’s scope to
support trials in other areas of NHSBT’s portfolio if required.
RECENT PUBLICATIONS
Massey E, Harding K, Kahan BC,
Llewelyn C, Wynn R, Moppett J,
Robinson SP, Green A, Lucas G,
Sadani D, Liakopolou E, BoltonMaggs P, Marks DI & Stanworth S.
The granulocytes in neutropenia 1
(GIN-1) study: a safety study of
granulocytes collected from whole
blood and stored in additive
solution and plasma. Transfusion
Medicine 2012; [Epub ahead of
print 16 May 2012 doi:10.111/
j.1365‑3148.2012.01152x ]
Muthukumar P, Venkatesh V,
Curley A, Kahan BC, Choo L,
Ballard S, Clarke P, Watts T,
Roberts I, Stanworth S,
for the Platelets Neonatal
Transfusion Study Group. Severe
thrombocytopenia and patterns
of bleeding in neonates: results
from a prospective observational
study and implications for use of
platelet transfusions. Transfusion
Medicine 2012; [Epub ahead of
print 27 Jun 2012 DOI:10.1111/
j.1365‑3148.2012.01171.x]
Walsh TS, Stanworth SJ, Prescott RJ,
Lee RJ, Watson DM, Wyncoll D;
Writing Committee of the Intensive
Care Study of Coagulopathy
(ISOC) Investigators. Prevalence,
management, and outcomes of
critically ill patients with prothrombin
time prolongation in United Kingdom
intensive care units. Crit Care Med.
2010 Oct;38 (10):1939-46
Research and Development Triennial Report 2009-12
25
Theme
1
3
Appropriate and safe use of blood components
Contribution to patient and donor care or service development
The granulocyte component assessed in the GIN-1 trial is now offered as a routine NHSBT
component.
The PRISM-A report was accepted by the Department of Health’s safety committee (SaBTO) as
part of the information needed to reach a final recommendation on implementation of the P-Capt
prion filter.
Theraflex (Jun 2010)
Evaluation of platelet
function in 12 platelet
donors following
pathogen inactivation
with u/v C
GIN (Nov 2010)
Safety of pooled blood
derived granulocytes in
additive solution and
plasma in 30 patients
with neutropenic sepsis
PPIP (Apr 2012)
Comparing in-vivo
efficacy of 2-5 day vs. 6-7
day platelets in 355
haemato-oncology
patients
HPA1a (Jan 2012)
Evaluation of modified
HPA1a antibody in 19
platelet donors
Key
Platelet trials
Red cell trials
CSU
Completed
Trials
2009 to 2012
TOPPS (Sep 2011)
Comparing policy of
prophylactic vs. non
prophylactic platelet
transfusions in 600
haemato-oncology
patients
Granulocyte trials
CSU Completed Trials 2009-12
26
Research and Development Triennial Report 2009-12
TAPS (Mar 2011)
Comparing policy of preoperative transfusion vs.
no transfusion in 70
sickle cell patients
PRISM-A (Apr 2011)
Safety study of prion
filtered red cells vs.
standard red cells in 590
transfused surgical
patients
Donor Palmwarming
(Jul 2011)
Evaluating venepuncture
related problems in 55
platelet-pheresis donors
given heated vs. unheated
palmwarmers
Theme
Appropriate and safe use of blood components 1
3
Components Development Laboratory
Dr Rebecca Cardigan
[email protected]
CONTRIBUTING TEAM MEMBERS: Stephen Thomas, Philip Cookson, Saber Bashir, Mike Wiltshire, Martin Beard,
Vicky Hancock, Sue Proffitt, Khairya Ishag, Lucy Backholer and Vijay Kaur
The Components Development Laboratory has three key roles:
1. to develop novel methods for producing blood components of improved quality
for transfusion
2. to evaluate commercial products designed to improve blood safety,
to ensure that product quality is not adversely affected, and
3. to advise operational departments on methods for quality monitoring.
Over the past three years the laboratory has focused on three areas:
1. filters to remove infectious prions (that transmit variant Creutzfeldt-Jakob Disease
vCJD) from red cells
2. systems to kill viruses and bacteria in platelets, plasma or red cells, and
3. the effect of short-term deviations from recommended storage temperature of
blood for transfusion.
We have undertaken two laboratory studies on systems to inactivate pathogens in
platelets and red cells. In collaboration with a commercial sponsor, we have assessed
an alternative system designed to inactivate pathogens in platelets by exposing
them to UVC light. We performed a clinical study in healthy volunteers to investigate
whether treating platelets in this way affects their ability to circulate once they have
been transfused. The sponsor is now proceeding to further clinical studies in patients.
NHSBT is frequently asked for advice on the likely impact of storing red cells or plasma
outside of their recommended storage temperature for short periods of time such
as may occur when equipment in blood centres or hospitals breaks down. There is a
lack of evidence on which to make informed decisions regarding whether affected
components are safe to transfuse, which led us to undertake a series of studies to
address this.
RECENT PUBLICATIONS
Thomas S, Hancock V, Cardigan R.
The 30-minute rule for red blood
cell concentrates – in vitro quality
assessment of red cells repeatedly
exposed to 30ºC. Transfusion 2012.
In press
Cookson P, Thomas S, Marschner S,
Goodrich R, Cardigan R. In vitro
quality of single-donor platelets
treated with riboflavin and ultraviolet
light and stored in platelet storage
medium for up to eight days.
Transfusion 2012; 52:983-94
Cardigan R, Themessl A, Garwood M.
Short-term Deviations in Temperature
During Storage of Plasma at -40ºC
Do Not Affect Its Quality. Transfusion;
51:1541-1545
Cardigan R, Van der Meer PF,
Pergande C, Cookson P, BaumannBaretti B, Cancelas JA, Devine D,
Gulliksson H, Vassallo R, de WildtEggen J. Coagulation factor content
of plasma produced from whole
blood stored for 24 hours at
ambient temperature: results from
an international multicenter BEST
Collaborative study. Transfusion 2011;
51 Suppl 1:50S-57S
We contributed to a systematic review of the literature to determine the maximal time
that a unit of red blood cells can be safely left out of controlled temperature storage,
as may occur when hospitals test it for compatibility with the patients blood, or to
transport them. Currently, units out of controlled temperature for 30 minutes are
discarded, on the basis of little evidence. This led to us to perform several studies to
assess the effect of storing red cells at room temperature on repeated occasions.
In conjunction with Professor Ouwehand’s group (NHSBT, University of Cambridge)
and the Clinical Studies Unit, we have recently started a clinical study to investigate
whether variation in the function of platelets in the blood donor population affects
their efficacy following transfusion. The study aim is to establish whether platelets
from some donors may be more beneficial for certain groups of patients.
Research and Development Triennial Report 2009-12
27
Theme
1
3
Appropriate and safe use of blood components
Components Development Laboratory
Contribution to patient and donor care or service development
Our work on prion reduction has led to new specifications being approved for
these components in the UK, in readiness to introduce this technology should it be
recommended to do so.
Our study on deviations in the normal storage temperature of plasma has been used
to develop guidance that can be used by hospitals/blood centres as part of their risk
assessment of whether affected units of plasma can be transfused.
We also have undertaken a review of the storage/thawing temperature for plasma
components. This resulted in a change in UK guidance to blood centres and hospitals
to allow greater flexibility.
28
Research and Development Triennial Report 2009-12
Theme
Erythrocyte biology and immunology 1
4
Making and manipulating red blood cells in
health and disease
Dr Ashley Toye
[email protected]
CONTRIBUTING TEAM MEMBERS: Mandy Bell, Alex Gampel, Bethan Hawley, Kathryn Mordue, Steph Pellegrin,
Timothy Satchwell and Charlotte Severn
It is particularly challenging for NHSBT to provide blood products to patients
who possess rare blood group types or for patients with multiple alloantibodies
due to frequent transfusions (eg patients with Sickle Cell or beta thalassemia).
One exciting solution is to culture red cells required for transfusion from
haematopoietic stem cells harvested from waste donor blood but further
optimisation is needed to generate the amounts of cells required for transfusion.
Importantly, whilst we further optimise the culture process we can use these
culture systems to study red cell development in health and disease.
To study the process of erythropoiesis, our group has established a 2D in vitro
erythroid culture system. We use CD34+ population or take all the stem cells present
in the peripheral blood mononuclear cell (PBMC) population to reproduce the
different stages of erythropoiesis from the proerythroblast to reticulocytes (van den
Akker et al, 2010).
Enucleating erythroblast stained
with RhAG monoclonal antibody
LA18.18 and the DNA stain DAPI.
Image provided by Mandy Bell,
University of Bristol.
We also use dexamethasone to delay terminal differentiation and then remove it
to allow erythroblasts to synchronously differentiate. We have used this culture
system alongside proteomics to monitor alterations in protein abundance (eg after
EPO withdrawal [Pellegrin et al, 2012]) and to investigate the processes behind
the assembly of the specialised red blood cell membrane in health and disease.
For example, we have compared the erythroid protein expression of erythroblasts
generated from a Hereditary Spherocytosis patient with that of a normal donor,
throughout terminal differentiation to the reticulocyte stage (van den Akker et al,
2010) identifying when characteristic alterations arise.
We also demonstrated that in normal donor erythroblasts the assembly of blood
group protein membrane complexes containing band 3 or Rhesus proteins begin
during the early (basophilic stage) of erythropoiesis (Satchwell et al, 2011) and these
key membrane proteins are delivered to the plasma membrane within 72 hours. This
expression profile means that designer blood cells are feasible and our laboratory is
now in the process of manipulating erythroid membrane protein expression.
In collaboration with researchers in Imperial College (Dr Anthanasios Mantalaris and
Dr Nicki Panoskaltsis), we are also exploring the use of bioengineered scaffolds to
mimic how the human body manufactures blood in the bone marrow. Recapitulating
the in vivo environment of the bone marrow niche should significantly reduce the cost
of red blood cell manufacture in the future, due to increased erythrocyte production
per stem cell input, and a reduction of both media volumes and dependence on
expensive cytokines.
Research and Development Triennial Report 2009-12
29
Theme
1
4
Erythrocyte biology and immunology
Contribution to patient and donor care or service development
A significant proportion of transfusion recipients have acute or chronic abnormalities
of erythropoiesis. Furthermore, for some patients with rare blood group types or for
patients with the presence of multiple alloantibodies due to frequent transfusions
(eg Sickle Cell or Beta Thalassemia) it is difficult to source appropriately matched
blood. To help address these issues, we are culturing haematopoietic stem cells from
donors, natural blood group variants and also from patients with a variety of red cell
diseases. This work will provide a better understanding of how red blood cells are
made, will also help us maximise red blood cell manufacture for bespoke erythrocytes
for transfusion and to improve our understanding about how this vital process can go
wrong in different human red blood cell diseases.
RECENT PUBLICATIONS
Pellegrin S, Heesom KJ, Satchwell TJ,
Hawley BR, Daniels G, et al, (2012)
Differential Proteomic Analysis of
Human Erythroblasts Undergoing
Apoptosis Induced by EpoWithdrawal. PLoS ONE 7(6): e38356.
Anstee DJ, Gampel A and Toye AM
(2012). Ex vivo generation of human
red cells for transfusion. Curr Opin
Hematol 19, 163-9
Satchwell TJ, Bell AJ, Pellegrin S,
Kupzig S, Ridgwell K, Daniels G,
Anstee DJ, van den Akker E and
Toye AM (2011) Critical band 3
multiprotein complex interactions
establish early during human
erythropoiesis. Blood 118, 182-91
van den Akker E, Satchwell TJ,
Pellegrin S, Flatt JF, Maigre M,
Daniels G, Delaunay J, Bruce LJ and
Toye AM (2010b) Investigating the
key membrane protein changes
during in vitro erythropoiesis of
protein 4.2 (-) cells (mutations
Chartres 1 and 2). Haematologica 95,
1278-86
Group webpage
http://www.bristol.ac.uk/
biochemistry/research/at.html
Scanning electron microscope image of a polyurethane
scaffold containing peripheral blood haematopoietic stem cells.
Image provided by Dr Alex Gampel, University of Bristol.
30
Research and Development Triennial Report 2009-12
Theme
Erythrocyte biology and immunology 1
4
Structure and function of red blood cells
Professor David Anstee
[email protected]
CONTRIBUTING TEAM MEMBERS: SF Parsons, LJ Bruce, BK Singleton, TJ Mankelow, RE Griffiths, NM Burton,
S Taylor (Kupzig), N Cogan, FA Spring and JF Flatt
Red blood cells are essential for life because they are the body’s vehicle for
transporting oxygen from the lungs to the tissues and carbon dioxide from the
tissues to the lungs. Consequently, providing anaemic patients with red blood
concentrates prepared from blood donations is a major function of NHSBT.
Our work aims to further understanding of how red cells are made and how they
function in order to develop new and improved red cell products for patients and to
elucidate the underlying causes of disease in patients with red cell disorders.
In the past three years we have developed a method for generating red cells in the
laboratory from stem cells, found in the waste products of normal blood donations, and
shown that these very young red cells have normal function. These results mean that red
cells grown in culture could be used for patient treatment in the future if we can expand
the numbers of red cells we grow. To do this we need to understand more about the
proteins involved in regulation of red cell production.
Rare form of anaemia
We were the first to identify genetic mutations in the human gene (EKLF/KLF1) encoding
a protein essential for the regulation of red cell production. We showed that most
mutations are benign but are associated with slightly raised levels of the fetal type of
haemoglobin. However, one particular mutation causes a rare form of anaemia and very
high levels of fetal haemoglobin. Mutations in EKLF have since been reported in a subset
of patients with unexplained haemoglobin disorders.
Red cell disorders
In certain diseases (eg hereditary stomatocytosis), red cells are unusually permeable to
potassium and we have been investigating the reasons for this. We have found a number
of mutations in multi-spanning membrane proteins (SLC4A1, RHAG, SLC2A1). These
mutant proteins are expressed in the red cell membrane and increase permeability by
forming cation channels or by disrupting the integrity of the membrane.
Contribution to patient and donor care or service development
Our work contributes to patient care through the development of cultured red cells for
use as a new therapeutic red cell product and by identifying the causative mutations in
rare red cell diseases we provide a reliable and precise method of diagnosis for these
patients.
Figure 1. Showing mature red blood cells from
peripheral blood. sdCHC is one of the hereditary
stomatocytosis group of cation permeable diseases
and is caused by a Gly286 to Asp286 mutation in the
glucose transporter (SLC2A1). The mutation causes
the loss of stomatin (a protein involved in vesicle
formation) during reticulocyte maturation.
Figure 2. Enucleating
early red blood cell
(reticulocyte).
Green: Cell surface
marker glycophorin A;
Blue: Nucleus
RECENT PUBLICATIONS
Griffiths RE, Kupzig S, Cogan N,
Mankelow TJ, Betin VM,
Trakarnsanga K, Massey EJ, Lane JD,
Parsons SF, Anstee DJ. Maturing
reticulocytes internalise plasma
membrane in glycophorin A –
containing vesicles, which fuse with
autophagosomes prior to exocytosis.
Blood 2012 Jun 28; 119(26): 6296-306.
Epub 2012 Apr 6
Singleton BK, Lau W, Fairweather VS,
Burton NM, Wilson MC, Parsons SF,
Richardson BM, Trakarnsanga K,
Brady RL, Anstee DJ, Frayne J. Mutations
in the second zinc finger of human
EKLF reduce promoter affinity but give
rise to benign and disease phenotypes.
Blood 2011 Sep 15;118(11):3137-45.
Epub 2011 Jul 21
Flatt JF, Guizouarn H, Burton NM,
Borgese F, Tomlinson RJ, Forsyth RJ,
Baldwin SA, Levinson BE, Quittet P,
Aguilar-Martinez P, Delaunay J,
Stewart GW, Bruce LJ. Stomatindeficient cryohydrocytosis results
from mutations in SLC2A1: a novel
form of GLUT 1 deficiency syndrome.
Blood 2011 Nov 10;118(19):5267-77.
Epub 2011 Jul 26
Giardine B, Borg J, Higgs DR,
Peterson KR, Philipsen S, Maglott D,
Singleton BK, Anstee DJ, Basak AN,
Clark B, Costa FC, Faustino P,
Fedosyuk H, Felice AE, Francina A,
Galanello R, Gallivan MV, Georgitsi M,
Gibbons RJ, Giordano PC,
Harteveld CL, Hoyer JD, Jarvis M,
Joly P, Kanavakis E, Kollia P, Menzel S,
Miller W, Moradkhani K, Old J,
Papachatzopoulou A, Papadakis MN,
Papadopoulos P, Pavlovic S, Perseu L,
Radmilovic M, Riemer C, Satta S,
Schrijver I, Stojiljkovic M, Thein SL,
Traeger-Synodinos J, Tully R, Wada T,
Waye JS, Wiemann C, Zukic B, Chui DH,
Wajcman H, Hardison RC, Patrinos GP.
Systematic documentation and
analysis of human genetic variation
in hemoglobinopathies using the
microattribution approach. Nat Genet.
2011 Mar 20;43(4):295-301
Group webpage
http://ibgrl.blood.co.uk
Research and Development Triennial Report 2009-12
31
Theme
1
4
Erythrocyte biology and immunology
Erythropoiesis in health and disease
Professor David Roberts
[email protected]
CONTRIBUTING TEAM MEMBERS: Abigail Lamikanra, Alison Merryweather-Clarke, Hoi Pat Tsang, Asoke Nandi,
Suzanne Watt, Lee Carpenter, Yang-Cheng Tao, Jackie Boultwood, Andrea Pellagati and Oscar Bon Yip
There is a need for alternative novel sources of red blood cells both for
therapy and for diagnostic use, where cells from individuals with uncommon
combinations of red cell antigens can be used in panels of cells with rare blood
groups for antibody testing. The expansion of red blood cells in the laboratory
can help to address these needs. Improving the expansion and differentiation of
red cells in the laboratory and in patients who have bone marrow disorders needs
a detailed understanding of cellular biology and gene expression during red cell
development (erythropoiesis) in healthy individuals and during disease.
We have previously identified many novel patterns of gene expression during
erythropoiesis in culture. Discrete populations of red cell precursors were isolated
based on identification of cellular markers. Each population represented one of
four different stages of erythropoiesis. This allowed precise identification of genes
expressed at each stage of erythropoiesis and identification of more genes whose
expression changed during development of red cells than previously observed in less
well defined mixed populations of precursors1 (Figure 1). Alongside this work, new
computational tools have been developed to validate gene expression data2. These
tools are now being applied to studying gene expression in red cell precursors derived
from cord blood, peripheral blood and human induced pluripotent stem cells (iPS).
Human iPS can be derived from the fibroblasts or blood of donors with rare blood
antigens to provide reagents for screening panels used in diagnostic laboratories. We
have optimised production of haematopoietic progenitors from human iPS cells from
fibroblasts3. The haematopoietic progenitors derived from these have been used to
generate red cell precursors that express a range of red cell antigens that indicate their
potential for use in diagnostic laboratories (Figure 2).
We are also studying defective erythropoiesis in red cells grown from the bone
marrow cells of patients with 5q- myelodysplastic syndrome (MDS). We have
identified a defect in the cellular machinery that produces functional proteins
from messenger RNA. The addition of L-leucine to these red cells grown in culture
reduced the characteristics associated with MDS highlighting its potential for use in
approaches to specifically treat 5q- MDS4. Taken together this work, funded by a
National Institute for Health Research (NIHR) programme grant, will help to define
and improve red cell development in the laboratory and we hope improve treatment
for patients with red cells disorders.
Contribution to patient and donor care or service development
The clinical need for alternative novel sources of red blood cells is considerable, not
only for patients who are difficult to cross match and require repeated transfusion but
also where rare groups are required for specific diagnostic applications. Improvement
of current methods used to produce red cells in the laboratory will be aided by a
detailed understanding of gene expression changes during erythropoiesis. This
knowledge may also lead us to novel treatments for people suffering from anaemia
due to abnormal development of red blood cells in the bone marrow.
32
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Merryweather-Clarke AT,
Atzberger A, Soneji S, Gray N,
Clark K, Waugh C, McGowan SJ,
Taylor S, Nandi AK, Wood WG,
Roberts DJ, Higgs DR, Buckle VJ,
Robson KJ. Global gene expression
analysis of human erythroid
progenitors. Blood. 117:e96-108
(2011)
1
Fa R and Nandi AK. Parametric
validity index of clustering for
microarray gene expression data, in
IEEE Int. Workshop Machine Learning
for Sig. Process 2011 (MLSP 2011),
2011
2
Carpenter L, Malladi R, Yang CT,
French A, Pilkington KJ, Forsey RW,
Sloane-Stanley J, Silk KM, Davies TJ,
Fairchild PJ, Enver T, Watt SM. Human
induced pluripotent stem cells are
capable of B-cell lymphopoiesis.
Blood 2011 Apr 14;117(15):4008-11
3
Yip BH, Pellagatti A, Vuppusetty C,
Giagounidis A, Germing U,
Lamikanra AA, Roberts DJ,
Fernandez-Mercado M, McDonald EJ,
Killick S, Wainscoat JS, Boultwood J.
Effects of L-leucine in RPS14-deficient
erythroid cells. Leukaemia 2012
Mar 20: 1-4
4
Figure 1
Theme
Erythrocyte biology and immunology A.
1
4
B.
Figure 1. Expression dynamics of the most significantly regulated transcripts during erythropoiesis.
A FIve-way Venn diagram of the 327 most significantly differentially expressed genes showing the overlap of fold change
combinations between stages of erythropoiesis: Late-E (L) and one of CFU-E (C, green), Pro-E (P, blue), or Int-E (I, red).
The wide magenta arc represents genes differentially regulated between Pro-Es and Int-Es. The narrower yellow arc represents
genes differentially regulated between CFU-E and Int-E. The colours merge where the shapes intersect each other.
B Five-way Venn diagram of the 327 most significantly differentially expressed transcripts showing the overlap of fold
combinations (see panel A). The white numbers represent the number of up-regulated genes whilst the black numbers
represent the number of genes that are down regulated.
Figure 2. Expression of Red Cell Antigens on Erythroid Cells Derived from iPS.
The expression of antigens in red cell precursors derived from human iPSC (iPSC) or from human adult stem cells (CD34)
was compared with expression of the respective antigens seen in mature red cells (RBC).
Research and Development Triennial Report 2009-12
33
Theme
1
4
Erythrocyte biology and immunology
Red cell diagnostics
Professor Marion Scott
[email protected]
CONTRIBUTING TEAM MEMBERS: Geoff Daniels, Jacky Gilmour, Kay Ridgwell, Rosey Mushens, Matt Burden,
Vanya Crew, Jonathan Dixey, Niamh Durcan, Kirstin Finning, Carole Green, Frances Green, Alan Guest, John Hosken,
Matt Hazell, Becky Lewis, Louise Tilley, Sue Tovey, Piers Walser, Marcin Wozniak and Laura Barry
Blood group phenotyping and pre-tranfusion cross-matching has for over
a century relied almost entirely on serological methodology. Now the
determination of the molecular basis of the major clinically significant blood
groups has paved the way for the introduction of novel techniques for pretransfusion compatibility testing.
We have carried out a large scale trial of commercially available genotyping
systems, and are carrying out research into new sequence based methods for
typing. Molecular biology technology has also permitted the production of novel
recombinant blood group antigens and antibodies to improve the detection of blood
group antibodies and antigens. We have also been working on the growth of red
cells from their precursors in the lab, to produce ‘designer’ reagent red cells for the
detection and identification of antibodies.
We have evaluated three commercially available blood group genotyping platforms
using a panel of over 1,000 DNA samples from NHSBT blood donors of known
extended phenotype. In all cases, the serologically determined phenotype concurred
with the phenotype predicted by the commercial genotyping platforms.
Further developments in technology
These low to medium throughput systems are thus currently suitable for extended
minor blood group genotyping of selected groups of donors and patients, and are
in process of being implemented in NHSBT. Routine high throughput blood group
typing of large numbers or all donors and patients would allow the safe allocation
of blood without further compatibility testing, by full electronic cross-matching. We
are currently exploring further developments in technology, such as next generation
pooled sequencing, to see if we can achieve this goal.
We are working with a commercial diagnostics supplier to develop recombinant red
cell antigens and antibodies for use in novel technology systems for the detection
and quantitation of antibodies and antigens. This partnership approach has led to the
accelerated development of these molecules, and the demonstration that they can
provide reliable sensitive systems for clinical use.
We have shown that we can grow red cells from precursor cells in the laboratory for
use as specialist ‘designer’ reagent red cells. We have shown that we can scale-up
production of these cells for diagnostic use, and that they perform well in a range of
serological techniques.
We have also been working on improvements to existing red cell diagnostics, and
have developed a technique for the accurate quantitation of antibodies during
pregnancy, using flow cytometry.
34
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Wozniak MJ, Bowring C, Lucas G,
Ridgwell K. Detection of HNA-3a and
3b antibodies using transfected cell
lines and recombinant proteins.
Transfusion 2012 Jul;52: 1458-1467
Volume 52, Issue 7, July 2012,
Pages: 1458–1467
Tilley L, Green C, Poole J, Gaskell A,
Ridgwell K, Burton NM, Uchikawa M,
Tsuneyama H, Ogasawara K,
Akkøk CA, Daniels G. A new blood
group system, RHAG: three antigens
resulting from amino acid
substitutions in the Rh-associated
glycoprotein. Vox Sang 2010
Feb;98(2):151-9
Daniels G, Finning K, Martin P,
Massey E. Non-invasive prenatal
diagnosis of fetal blood group
phenotypes: current practice and
future prospects. Prenat Diagn.
2009; 29(2):101-7
Group webpage
http://IBGRL.blood.co.uk/
research/Prog3/Prog3-frames.htm
Theme
Erythrocyte biology and immunology 1
4
Contribution to patient and donor care or service development
All RhD-negative pregnant women are currently offered treatment with anti-RhD
immunoglobulin during pregnancy to prevent antenatal RhD immunisation and
subsequent potential haemolytic disease of the foetus and new-born. However,
about 40% of these women are carrying a RhD-negative fetus, and so receive the
treatment unnecessarily. We have developed a high throughput automated method
of determining fetal RhD type from maternal plasma. This has the potential to reduce
the wastage and cost of anti-RhD immunoglobulin and also save 40,000 pregnant
women per year from unnecessary treatment with a pooled blood product. A
regional pilot trial is currently underway.
Research and Development Triennial Report 2009-12
35
Theme
1
5
Platelet biology and genomics
Placing signposts in the genome for the formation
and function of platelets
Professor Willem Ouwehand
[email protected]
CONTRIBUTING TEAM MEMBERS: Cornelis Albers, Ana Cvejic, Nicola Foad, Stephen Garner, Augusto Rendon,
Jennifer Sambrook and Peter Smethurst
The focus of the genomics programme is on one type of blood cell, named the
platelet. Since their discovery in 1841 as the “Minute Spherules about 1/10,000 of
an Inch”, scientists have largely remained incognisant of the genes that regulate
their formation. An understanding of the molecular mechanisms that control the
mass of platelets and their pro-thrombotic function is relevant for diseases like heart
attacks and stroke, the number one killer. Additionally it will aid studies to generate
platelets from pluripotent stem cells either to replace classic donor-derived platelet
concentrates or to generate platelets with specific functional features, ie for vessel
wall repair from gene-engineered stem cells (see Ghevaert, page 42).
In the 2009 Report we described the use of a Genome Wide Association Scan (GWAS)
to identify the first four genes that regulate the count and volume of platelets. A global
collaboration by the HaemGen consortium (co-leads Dr Christian Gieger, Prof Willem
H Ouwehand and Dr Nicole Soranzo) expanded the GWAS to nearly 67,000 healthy
individuals and identified another 64 independent association signals1. Nearly all sentinel
SNPs (the ones with lowest P-value of association) were localised within 10kb of a
gene allowing inference of gene candidacy. The functional roles in megakaryopoiesis
and platelet formation of the vast majority of these genes are unknown. To close this
knowledge gap we silenced genes in zebrafish and profound effects on the formation
of thrombocytes were observed for 23 of the 27 genes investigated so far (see Cvejic,
page 40).
With the GWAS era coming to an end, studies have re-focussed on the discovery of
genes that underlie rare platelet bleeding disorders. Next generation sequencing made
it feasible to survey the ~64Mb coding fraction of the genome or so called exome. The
exome sequencing of four Grey Platelet Syndrome cases and six with Thrombocytopenia
and Absent Radii (TAR) identified NBEAL22 and RBM8A3 as causative genes,
respectively. The former was a classic example of autosomal recessive coding mutations,
but the genetic architecture of TAR is more complex. A deletion on 1q21.1 on one
haplotype is typically accompanied by a 5’ UTR SNP in the RBM8A gene.
RECENT PUBLICATIONS
1
Gieger C*, Radhakrishnan A*,
Cvejic A*, et al, (2011). New gene
functions in megakaryopoiesis and
platelet formation. Nature, 480,
201–208
2
Albers CA*, Cvejic A*, Favier R, et al,
(2011). Exome sequencing identifies
NBEAL2 as the causative gene for Gray
Platelet Syndrome. Nat Genet, 43,
735–737
Albers CA*, Paul DS*, Schulze H*,
et al, (2012). Inheritance of lowfrequency regulatory SNPs and a rare
null mutation in exon-junction complex
subunit RBM8A causes TAR. Nat Gen,
44, 435–439
3
4
Tijssen MR*, Cvejic A*, Joshi A, et al,
(2011). Genome-wide analysis of
simultaneous GATA1/2, RUNX1, FLI1,
and SCL binding in megakaryocytes
identifies hematopoietic regulators.
Developmental Cell, 20, 597-609
*authors contributed equally
Group webpage
http://www.haem.cam.ac.uk
The functional importance of this SNP was inferred from the annotation of the genomes in megakaryocytes4 and erythroblasts
(see Rendon, page 37). Genotyping in the Cambridge BioResource of 12,000 individuals (see Foad, page 11) showed a one in 30
frequency for the UTR-SNP with no apparent effect on platelet count in individuals homozygous for the minor allele.
Patients with a heart attack or after angioplasty receive two platelet-inhibiting drugs, sometimes even three, to reduce the chance
of recurrence of thrombus formation. Bleeding occurs as a side effect in ~1.5% of patients, often requiring transfusion of blood
and platelets. The use of anti-platelet drugs will be one of the first areas in which precision medicine will be pioneered and
implemented. To deliver this innovation we need to fully appreciate how the interaction between our genes and environment
shapes the mass of platelets and their function. For this translation to succeed a partnership between academia, health care
providers, pharmaceutical and biotechnology companies and regulators is required.
(A) Protein-protein interaction (PPI) network of platelet GWAS loci. 44 of the 67 core genes
connected through first-order interactions in a network containing 785 nodes and 1,085 edges.
For the nodes genes are represented by round symbols, where node colour reflects gene transcript
levels in megakaryocytes on a continuous scale from low (dark green) to high (white). Greycoloured round symbols identify first-order interactors identified in Reactome and IntAct. Core
genes not connected to the main network are omitted. The 34 core genes are identified by a blue
perimeter. Yellow perimeters identify five additional genes (VWF, PTPN11, PIK3CG, NFE2 and MYB)
with known role in haemostasis and megakaryopoiesis and mapping to within the association
signals at distances greater than 10kb from the sentinel SNPs. Network edges obtained from the
Reactome (blue) and IntAct-like (red) databases and through manual literature curation (black).
The full network, containing gene expression levels and other annotation features, is available in
Cytoscape format for download (see recent publications1).
36
Research and Development Triennial Report 2009-12
Platelet biology and genomics Theme
1
5
Functional genomics interpretation of the genetic
determinants of haematological parameters
Dr Augusto Rendon
[email protected]
CONTRIBUTING TEAM MEMBERS: Anthony Attwood, Stuart Meacham, John Ord, Aparna Radhakrishnan
and Yagnesh Umrania
The application of statistical genomics and computational biology has become essential in blood cell biology research.
Genome wide association scans (GWAS) carried out by our collaborators and ourselves have led to the identification of
nearly 150 regions of the genome, many of which were previously unknown, where common differences in the DNA
sequence result in changes in the way red cells and platelets are produced.
GWAS has achieved great success at identifying areas of the genome associated with the size and number of these cells; however,
because of the correlation between DNA variants – linkage disequilibrium – the actual genes and genetic mechanisms responsible
for the association cannot be readily inferred.
To overcome this limitation, we have functionally annotated the genomes of the progenitor cells of the red cells and platelets
(erythroblasts and megakaryocytes, respectively). In particular, we have traced the expression of all genes by whole genome
expression arrays as these two cells differentiate from their haematopoietic progenitors. For end-stage differentiated erythroblasts
and megakaryocytes, we have also mapped genome-wide regions of open chromatin, various other epigenetic marks and
transcription factor binding sites, as well as sequenced their RNA.
This functional annotation has allowed us to identify putative causative genes, two thirds of which are novel regulators of
erythropoiesis and megakaryopoiesis and for which we have provided experimental validation in model organisms (see Cvejic,
page 40). By analysing the expression patterns of these genes we have also concluded that common sequence variation affect
genes active during the late stages of differentiation in a lineage-specific manner rather than at the megakaryocyte-erythroblast
progenitor cell level. For two genomic regions at 1q24.3 (DNM3) and 7q22.3 (PIK3CG), we have also defined the genetic
mechanisms underlying the observed association between common sequence variants at these loci and the volume of platelets.
GWAS have the unparalleled advantage of providing a broad and unbiased catalogue of genes implicated in controlling the mass
of red cells and platelets, but this catalogue is incomplete. To increase our understanding of the molecular processes controlling
the formation of platelets from megakaryocytes, we have also developed a high quality protein-protein interaction network of
785 nodes (proteins) and 1,085 edges (pathway reactions) by conservatively expanding our list of known regulators with first
order interacting proteins. This has provided an unprecedented picture of the molecular architecture of the formation of this
blood cell type.
Contribution to patient and donor care or service development
The functional annotation of the genome of erythroblasts and megakaryocytes ultimately will provide a greater insight in the
regulation of gene transcription, which drives the fating events at branch points during differentiation of the haematopoietic
stem cell. The integration of this information by means of computational biology and statistical genomics is essential in defining
the molecular mechanisms by which GWAS-identified genes control the formation of red cells and platelets. The knowledge about
these molecular mechanisms is critical for the development of methods to generate both blood cell types from pluripotent stem
cells in the laboratory.
Research and Development Triennial Report 2009-12
37
Theme
1
5
Platelet biology and genomics
RECENT PUBLICATIONS
Soranzo N, et al. A genome-wide
meta-analysis identifies 22 loci
associated with eight hematological
parameters in the HaemGen
consortium. Nat Genet 41, 1182–1190
(2009)
Paul DS, et al. Maps of Open
Chromatin Guide the Functional
Follow-Up of Genome-Wide
Association Signals: Application to
Hematological Traits. PLoS Genet 7,
e1002139 (2011)
Gieger C, et al. New gene functions
in megakaryopoiesis and platelet
formation. Nature 480, 201–208
(2011)
Wang D, Rendon A, Ouwehand W
and Wernisch L. Transcription factor
co-localization patterns affect human
cell type-specific gene expression.
BMC Genomics 13, 263 (2012)
The functional annotation of the
DNM3 locus shown across three
panels:
A The DNM3 locus at 1q24.3 spans
about 500kb and the sentinel SNP
associated with platelet volume
at a P-value of 1.11 x 10 -24 as
identified by GWAS is rs109141441.
B Formaldehyde-Assisted
Isolation of Regulatory Elements
(FAIRE) shows regions of open
chromatin, with one at variant
rs2038479, which is high
Linkage Disequilibrium with
the sentinel SNP. Chromatin
immunoprecipitation combined
with massive parallel sequencing.
(ChIP-seq) showed that the latter
SNP marks a binding site for the
transcription factor MEIS1 and
the same 'intronic' element is
transcribed in megakaryocytes as
shown by RNA-seq.
C Variant rs2038479 marks an
alternative DNM3 promoter
encoding for a DNM3 molecule
which lacks its GTPase domain.
This alternative transcript is only
present in megakaryocytes and
not in other tissues in which the
DNM3 gene is transcribed.
38
Research and Development Triennial Report 2009-12
Theme
Platelet biology and genomics 1
5
A blueprint of haematopoietic epigenomes
Professor Willem Ouwehand
[email protected]
CONTRIBUTING TEAM MEMBERS: Mattia Frontini, Kate Downes, Daniel Hampshire, Frances Burden
and Samantha Farrow
The BLUEPRINT consortium has been formed with the aim to further the understanding of how genes are activated or
repressed in both healthy and diseased human cells with particular focus on distinct types of haematopoietic cells from
healthy individuals and on their malignant leukemic counterparts. It aims to generate at least 100 reference epigenomes
and study them to advance and exploit knowledge of the underlying biological processes and mechanisms in health
and disease. Reference epigenomes will be generated by state-of-the-art technologies from highly purified cells for a
comprehensive set of epigenetic marks in accordance with quality standards set by IHEC.
This resource-generating activity will be complemented by hypothesis-driven research into blood-based diseases, including
common leukaemia and autoimmune disease (Type 1 Diabetes), by discovery and validation of epigenetic markers for diagnostic
use and by epigenetic target identification. Since epigenetic changes are reversible, they can be targets for the development of
novel and more individualised medical treatments.
The Cambridge team has the task of purifying healthy cell types from NHSBT donors.
Contribution to patient and donor care or service development
Blueprint will generate a reference resource that will allow medical professionals worldwide to compare data generated from
patients with healthy individuals. Moreover defects in epigenetic regulation are emerging as one of the leading causes of many
diseases. Identification of key epigenetic factors and compounds able to inhibit or enhance their function are at the forefront of
personalised medicine.
Group webpage
http://www.blueprint-epigenome.eu
Research and Development Triennial Report 2009-12
39
Theme
1
5
Platelet biology and genomics
Elucidating the function of novel genes in
haematopoiesis in zebrafish
Dr Ana Cvejic PhD
[email protected]
CONTRIBUTING TEAM MEMBERS: Dana C Bellissimo, Ewa Bielczyk, Nicolas Brieu* and Jovana Serbanovic-Canic
*Computer Aided Medical Procedures, Technische University of Munich, Munich, Germany
Zebrafish as a model organism combines the advantages of both invertebrate
and vertebrate model systems. The genes orchestrating both haematopoiesis and
haemostasis are highly conserved providing a highly suitable model system to
rapidly investigate the function of novel genes.
Genomics studies (see Ouwehand, page 36) have identified a plethora of novel genes,
which are assumed to play critical, and pathway restricted roles in megakaryopoiesis
and the formation and function of platelets. The accurate placement of the proteins
encoded by these novel genes in the protein-protein interaction network, which
regulates these processes, requires experimental studies in model organisms, like
zebrafish.
These studies benefit from mathematical algorithms for the automated interpretation
of phenotype. To achieve this we collaborated with Prof Nassir Navab’s team at the
Technical University of Munich and developed a method to automatically interpret
thrombus formation after laser-induced vessel wall damage in a sensitive and specific
manner1.
In parallel, quantitative methods were validated to enumerate the number of
different blood cell elements, including haematopoietic stem cells and thrombocytes.
First, these methods were used to determine the role of meis1, during zebrafish
primitive and definitive haematopoiesis. Analysis of our compendium of transcripts
in mature human blood cell elements showed that the MEIS1 transcript was the
only transcription factor-encoding transcript abundantly present in megakaryocytes
but absent from others. Meis1 silencing by morpholino injection ablates erythroid
and thrombocyte formation2. In addition, it leads to dramatic single arteriovenous
tube formation. Nuclear entry of meis1 depends on its interaction with pbx1 and as
expected knockdown of pbx1 results in a strikingly similar phenotype to that of meis1
knockdown.
GWAS and ChIP-seq3 identified 68 and 151 genes, which putatively encode important
regulators of megakaryopoiesis and platelet formation. Two thirds of these encode
proteins of unknown function. To gain insight in their role, 17 were selected for
silencing in zebrafish and for all but three profound effects on both thrombopoiesis
and interestingly also on erythropoiesis were observed. Further studies on the
guanine nucleotide exchange factor arhgef3 showed its role in erythropoiesis through
activation of rhoa. Lentiviral mediated silencing in human K562 cells identified
ARHGEF3 as a new regulator of iron homeostasis. Finally, exome sequencing
revealed NBEAL2 as the causative gene of Grey Platelet Syndrome and silencing of its
homologue showed severe spontaneous bleeding in about 20% of the zebrafish.
Contribution to patient and donor care or service development
The studies in zebrafish in Dr Derek Stemple’s laboratory at the Wellcome Trust
Sanger Institute are essential in elucidating the function of novel genes. The
juxtaposition of these studies between the genomics efforts (Ouwehand, page 36)
and the programme on human pluripotent stem cells (Ghevaert, page 42) has
created a unique opportunity to transform our understanding of the intricacies of
the molecular regulation of platelet formation and function. This will eventually have
its bearing on the care of patients with heart attacks or stroke and the provision of
platelet concentrates by NHSBT for patient care.
40
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Brieu N, Navab N, SerbanovicCanic J, Ouwehand WH, Stemple DL,
Cvejic A*, Groher M*, (2012). Imagebased Characterization of Thrombus
Formation in Time-lapse DIC
Microscopy. Medical Image Analysis,
16(4):915-31. (*joint last authors)
1
Cvejic A, Serbanovic-Canic J,
Stemple DL, Ouwehand WH, (2011).
The role of meis1 in primitive
and definitive haematopoiesis
during zebrafish development.
Haematologica, 96(2): 190-198
2
Tijssen MR*, Cvejic A*, Hannah RL,
Ferreira R, Forrai A, Bellissimo DC,
Joshi A, Wilson NK, Wang X,
Ottersbach K, Stemple DL, Green AR,
Ouwehand WH, Göttgens B, (2011).
Genome-wide analysis of GATA1,
GATA2, RUNX1, FLI1 and SCL
binding in megakaryocytes identifies
8 new hematopoietic regulators.
Developmental Cell, 20(5):597-609.
(*joint first authors)
3
Serbanovic-Canic J*, Cvejic A*,
Soranzo N, Stemple DL,
Ouwehand WH, Freson K, (2011).
Silencing of RhoA nucleotide
exchange factor, ARHGEF3 reveals its
unexpected role in iron uptake. Blood,
118(18):4967-76. (*joint first authors)
Group webpage
http://www.haem.cam.ac.uk
Theme
Platelet biology and genomics 1
5
Platelet biology and quantitative trait loci
Stephen Garner and Dr Peter Smethurst
[email protected] and [email protected]
Stephen Garner
Dr Peter Smethurst
CONTRIBUTING TEAM MEMBERS: Abeer Al-Subaie, Abigail Crisp-Hihn, Sjoert Jansen,
Dr Anne Kelly*, Dr Sylvia Nürnberg and Dr Rafik Rizkallah (*Clinical Fellow supervised by Dr Rebecca Cardigan)
Platelets are the second most abundant cell in blood and are important in surveying
the arterial vessel wall for damage and to initiate repair. A very low number, or
poorly functioning platelets may lead to bleeding (see Rebecca Cardigan page 27
and Simon Stanworth page 22). It is therefore attractive to postulate that having
a high platelet mass (count x volume) and functionally active platelets has been
evolutionarily advantageous. With today’s sedentary lifestyle having these two
characteristics may be less beneficial because both are independent risk factors for
stroke and heart attacks. The pro-thrombotic function of platelets varies widely in
the healthy population and is strongly heritable, as are platelet count and volume.
The principal purpose of our research is to identify sequence variants that regulate
the platelet functional response to activation.
To support this endeavour and to provide well-characterised donors for the Platelet
Responsiveness and Outcome from Platelet Transfusion (PROMPT) study (see Simon
Stanworth, page 22) the Cambridge Platelet Function Cohort was expanded from
500 to 1,000 donors. The functional response of their platelets upon activation with
adenosine 5’-diphosphate or both the collagen mimetic CRP-XL and the membrane
expression levels of both collagen receptors (glycoprotein VI and α2 β1 integrin) and the
fibrinogen receptor (α1b β3 integrin) were measured by flow cytometry. Platelet RNA
samples from donors representing the full range of functional responses were applied
to whole-genome expression arrays, and analysis of this microarray data identified 63
transcript levels that correlated with variation in platelet functional response.
We wished to determine whether the corresponding genes were important regulators
of thrombus formation. COMMD7 and LRRFIP1 were selected for further study based on
the observation that common sequence variants in both loci seemed to be associated
with the risk of heart attacks in 4,235 cases of premature myocardial infarction
compared with 6,379 controls. A zebrafish model of arterial thrombus formation was
developed and silencing of both commd7 and lrrfip1 genes showed reduced thrombus
size upon laser-wounding of the vessel wall identifying both proteins as novel positive
regulators of thrombus formation.
RECENT PUBLICATIONS
Goodall AH, Burns, P Salles I, et al,
(2010). Transcription profiling in
human platelets reveals LRRFIP1 as
a novel protein regulating platelet
function. Blood. 116:4646-56
Soranzo N, Rendon A, Gieger C,
et al, (2009). A novel variant on
chromosome 7q22.3 associated with
mean platelet volume, counts, and
function. Blood. 113:3831-7
Jones CI, Garner SF, Moraes LA,
et al, (2009). PECAM-1 expression and
activity negatively regulate multiple
platelet signalling pathways. FEBS Lett,
583, 3618-3624
Peter L, Brieu N, Jansen S, et al,
(2012). Automatic segmentation and
tracking of thrombus formation within
in vitro microscopic video sequences.
Proceedings of the International
Symposium on Biomedical Imaging
(in press)
Group webpage
http://www.haem.cam.ac.uk
In order to study how dynamic thrombus formation differs in human blood samples we
have subsequently established a novel microscope based image processing method.
The Platelet Function Cohort has also been used for in silico association studies for other
genes identified by the Genome Wide Association Scan (GWAS) for platelet count and
volume. This revealed that common sequence variation at Chromosome 7q22.3 exerts not
only an effect on the volume of platelets but also on their function.
Contribution to patient and donor care or service development
The Cambridge Platelet Function Cohort is an international reference for platelet function
variation. This will become important for care of patients after a heart attack or an
angioplasty. These patients receive two or three platelet inhibitors to prevent thrombus
formation, but this is associated with a risk of severe bleeding. It is hoped future treatment
with anti-platelet drugs will be more precise and be informed by knowledge of the
patient’s platelet function status. Whether platelet concentrates prepared from donors
with highly active platelets are clinically less effective is being addressed in the PROMPT
study (see page 22).
Small and large thrombi may be
visualized by microscopy. Video
analysis enables the start and
growth of individual thrombi
to be measured over time, as
shown by the different coloured
lines in the graph.
Research and Development Triennial Report 2009-12
41
Theme
1
5
Platelet biology and genomics
In Vitro production of platelets for transfusion
Dr Cedric Ghevaert
[email protected]
CONTRIBUTING TEAM MEMBERS: Dr Thomas Moreau and Dr Meera Arumugam
Each year the NHSBT produces a quarter of a million of platelet concentrates for
transfusion to patients who would otherwise develop severe bleeding without
blood product support as a consequence of cancer therapy, trauma or surgery.
Fifteen thousand of these units have to be sourced from specially matched
donors for patients who are refractory to the standard platelet concentrates. This
represents a significant logistical and financial burden for the NHSBT and the NHS
as a whole.
My group is concentrating its effort on producing platelets and megakaryocytes
(the cell in the bone marrow from which platelets are made) from stem cells. Our
goal in the long term is to establish banks of stem cells and a production method for
platelets, which would cover over 80% of the matched platelet demand in the UK.
Over the last two years we have worked on an efficient and novel way to promote
megakaryocyte growth from stem cells and have now reached a point where we
obtain reliably large numbers of megakaryocytes for validation and pre-clinical testing
in conditions that are compatible with the necessary standards for human therapeutic
products.
Figure 1. Megakaryocytes grown in
vitro from induced pluripotent stem
cells. The morphology of the cells
with large amount of cytoplasm and,
in some cases, multiple nuclei in one
cell is entirely consistent with the
megakaryocytes found in the bone
marrow.
Our approach is based on the forward programming of the stem cells towards
megakaryocytes by the exogenous expression of proteins that are key regulators
of megakaryocyte growth called ‘transcription factors’. Transcription factors are
numerous and form networks of association that control whether a gene is ‘on’ or
‘off’. Through genetic studies, basic research of transcription factors binding to genes
in megakaryocytes and comparison of transcription factors at play in stem cells and
megakaryocytes we identified key proteins that are able to make the switch from
stem cells to megakaryocytes. This discovery has now been patented (GB1210857.7).
Alongside this work, we have developed three-dimensional scaffolds in collaboration
with engineers from the University of Cambridge onto which we will seed the stem
cells in a bid to scale-up production in bioreactors and provide sufficient numbers
of cells for clinical applications. One crucial aspect of the scaffold is that it can be
modified so as to give a signal for the megakaryocytes to produce platelets efficiently
that can be harvested for transfusion into patients. Both lines of research are
groundbreaking and integral to our goal to develop an alternative way to generate
platelets that are safe, efficient and matched to patients which will translate in
adequate prevention of bleeding, reduced hospital stay and gain in quality of life.
Contribution to patient and donor care or service development
The provision of platelets, especially HLA-matched platelets for patient refractory to
standard platelet pools is a huge logistical and financial burden to the NHS. We are
seeking an alternative way to provide platelets for transfusion by producing them
in vitro using a source of stem cells that have unlimited maintenance and expansion
potential called human pluripotent stem cells. Although still in the early phase, our
research shows that this approach has the potential in the future to provide a blood
product that is safe, efficient and addresses some of the immunological issues around
platelet transfusion by providing matched products for patients who are refractory to
the standard donor-derived platelets.
42
Research and Development Triennial Report 2009-12
Figure 2. Scanning electron microscopy
image of a 3D collagen scaffold showing
the porous structure and niche into
which the megakaryocyte can grow
whilst allowing circulation of nutrient
and the collection of platelets shed by
the mature megakaryocytes.
Theme
Platelet biology and genomics 1
5
A study of the HPA-1a Antibody Response in
Neonatal Alloimmune Thrombocytopenia (NAIT)
Professor David Roberts
[email protected]
CONTRIBUTING TEAM MEMBER: David Allen
Neonatal alloimmune thrombocytopenia (NAIT) occurs as a result of maternal
antibodies against proteins present on fetal platelets. These antibodies can result in
the destruction of the fetal or neonatal platelets and bleeding which may include
brain or intracranial haemorrhage so leading to disability or death. Current tests for
these antibodies do not reliably predict the severity of disease making screening and
treatment of affected women and children difficult. We have studied the nature and
characteristics of antibodies in this condition and aim to devise better tests to prevent
and manage this condition.
The most frequently encountered antibody is known as HPA-1a and it recognises a
particular form of a protein known as β3. This protein is found on platelets as part of a
larger protein complex (αIIbβ3) and tests to detect HPA 1a antibody use this protein as
a target antigen.
We have investigated whether assays used in the laboratory to detect HPA–1a
antibodies actually cause changes to the conformation of the protein and prevent their
detection. We have also studied the use of these different forms αIIbβ3 can improve
the sensitivity and predictive ability of the test.
We have shown that changes on the αIIbβ3 protein may be brought about by the type
of anticoagulant used for collection of maternal blood and also by reagents used in
diagnostic assays. We have also shown potential structural differences of this integrin
under different assays conditions. All of these findings can affect how well the maternal
antibodies are detected and also how much maternal antibody appears to be present.
RECENT PUBLICATIONS
Allen DL, Abrahamsson S, Murphy MF
and Roberts DJ, (2012). Human
platelet antigen 1a epitopes are
dependent on the cation-regulated
conformation of integrin alpha(IIb)
beta(3) (GPIIb/IIIa). J. Immunol
Methods, 375, 166-175
Knight M, Pierce M, Allen D,
Kurinczuk JJ, Spark P, Roberts DJ and
Murphy MF, (2011). The incidence and
outcomes of fetomaternal alloimmune
thrombocytopenia: a UK national
study using three data sources.
Br J Haematol, 152, 460-468
Metcalfe P, Allen D, Kekomaki R,
Kaplan C, De HM and Ouwehand WH,
(2009). An International Reference
Reagent (minimum sensitivity) for
the detection of anti-human platelet
antigen 1a. Vox Sang, 96, 146-152
We now need to evaluate whether new assays, developed as a result of these findings,
are better able to detect and measure maternal antibodies and are therefore better able to predict NAIT. Ideally this should be
done in a large prospective study where sequential samples have been taken from a large cohort of HPA-1b1b women during
their pregnancy and where there is data on the clinical outcome and platelet count in neonates. We would now like to test our
modified assay in parallel with existing and other novel techniques, including the recombinant β3 proteins to determine the
relative sensitivity and specificity of these assays. We could then take the most suitable assay forward into a large scale trial of
screening for HPA-1a antibodies. We would hope such a trial would define the value of antenatal screening to predict which
children might be affected and provide treatment before they become severely affected.
Contribution to patient and donor care
Our findings provide possible explanations for the reported insensitivity of current tests for platelet antibodies and why it has
not been possible to predict with certainty which children will be affected by NAIT. We have shown that key reagents used
in diagnostic tests have an impact on antibody detection and some of these techniques have been adopted by laboratories
around the world that provide these diagnostic services. Future work will define how a new test for HPA-1a antibodies can
improve the prediction of severe disease.
(A) Molecular models of αIIbβ3 protein. The HPA-1a epitope lies within the circled area and is shaded
black. The lower figure is a magnified view of the circled area.
Research and Development Triennial Report 2009-12
43
Theme
1
5
Platelet biology and genomics
Recombinant antibody for treatment of fetomaternal
alloimmune thrombocytopenia
Dr Cedric Ghevaert
[email protected]
CONTRIBUTING TEAM MEMBERS: Louise Hawkins, Nina Herbert, Paul Lloyd-Evans, Phil Cookson and
Dr Lorna Williamson
In the UK, one in 350 pregnant women will develop antibodies against their baby’s
platelets. One in three babies will see their platelet count drop significantly as a
result of these maternal antibodies crossing the placenta into the baby’s circulation
and binding to the baby’s platelets. As a consequence of this drop in platelet count
the babies can develop severe bleeding, including in the brain, which can lead to
life-long disability or even death in the womb.
Antenatal therapy is usually with a combination of immunosupressors and intravenous
immunoglobulins taken by the pregnant mother. Treatment at the time of birth is with
transfusion of platelets that do not carry the antigen to which the maternal antibodies
bind. Despite therapy some intracranial bleeds still happen and side effects of the
treatment itself for the pregnant mother should also be considered.
We have taken the approach of developing an antibody that is similar to the mother’s
and therefore can compete with maternal antibodies for binding to the baby’s platelets.
However we have modified our therapeutic antibody so that it does not activate the
destruction of the platelets it is bound to. In essence our new compound would prevent
platelet destruction in the baby by the maternal antibodies with the added benefit of
being generated in the laboratory and therefore a much better side-effect profile than
current treatments.
‘First-in-man’ study
Previous work in the laboratory confirmed the validity of our approach. We have now
carried out a ‘first-in-man’ study in healthy volunteers. These volunteers were recruited
from an NHSBT donor panels known to match the babies’ blood group. A blood sample
taken from the volunteer allowed us to generate platelets that were coated with either
a destructive antibody (similar to the maternal antibody) or with our new compound.
These platelets were re-injected into our volunteers and their lifespan in the circulation
was assessed by means of a radioactive label attached to the platelets.
We found that survival of platelets coated with the therapeutic antibody was far
superior to the survival of platelets coated with the destructive one and was in fact as
good as platelets not coated with any antibody at all. We are now therefore looking at
the possibility of taking this potential treatment into clinical trials in collaboration with
other researchers across Europe and partners in the pharmaceutical industry.
Contribution to patient and donor care or service development
RECENT PUBLICATIONS
Ghevaert C, Wilcox DA, Fang J,
Armour KL, Clark MR, Ouwehand
WH, et al. Developing recombinant
HPA-1a-specific antibodies with
abrogated Fcgamma receptor binding
for the treatment of fetomaternal
alloimmune thrombocytopenia. The
Journal of clinical investigation. 2008
Aug;118(8):2929-38
Ghevaert C, Campbell K, Walton J,
Smith GA, Allen D, Williamson LM,
et al. Management and outcome of
200 cases of fetomaternal alloimmune
thrombocytopenia. Transfusion. 2007
May;47(5):901-10
Ghevaert C, Rankin A, Huiskes E,
Porcelijn L, Javela K, Kekomaki R, et al.
Alloantibodies against low-frequency
human platelet antigens do not
account for a significant proportion
of cases of fetomaternal alloimmune
thrombocytopenia: evidence from 1,054
cases. Transfusion. 2009 Jun 4. 2009
49;2084-9
Ghevaert C, Campbell K, Stafford P,
Metcalfe P, Casbard A, Smith GA, et al.
HPA-1a antibody potency and bioactivity
do not predict severity of fetomaternal
alloimmune thrombocytopenia.
Transfusion. 2007 Jul;47(7):1296-305
Group webpage
http://www.haem.cam.ac.uk/staff/
senior-staff/cedric-ghevaert
Fetomaternal alloimmune thrombocytopenia (FMAIT) can be a devastating disease
leading to either death of fetuses in the womb or to bleeding in the baby’s brain and
long-term disability.
The NHSBT provides support for hospitals dealing with cases with FMAIT in two ways: 1.
Investigating for the presence of antibodies in the maternal blood responsible for FMAIT
and 2. Providing antigen-negative platelet support for transfusion to the affected fetuses
and neonates. The development and ‘first-in-man’ trial of a new therapeutic compound
to prevent destruction of the platelets will potentially change completely our treatment
approach for this disease and the logistics of providing matched platelets for treatment
of affected babies.
Figure 1. We have produced a therapeutic antibody (in green) that can be administered to a pregnant mother who has herself formed
antibodies against her unborn baby’s platelets (in red). The therapeutic antibody can be given by injection to the mum, crosses the placenta
and outcompetes the maternal antibodies for binding to the baby’s platelets. Once bound to the platelets, the therapeutic antibody blocks
platelet destruction by the effector cells, thereby reducing the risk of bleeding in the baby.
44
Research and Development Triennial Report 2009-12
Theme
Organ donation and transplantation 1
6
Quality in organ donation (QUOD)
Professor Rutger J Ploeg
Principal Investigators: John Dark, Peter Friend, Anthony Gordon, Gerlinde Mandersloot, Wayel Jassem,
Lorna Marson and Christopher Watson
Expert Advisors: Christian Brailsford, David Collett and Sue Fuggle
Research Students: Zeeshan Akhtar, Maria Kaisar and Rachel Thomas
One of the biggest challenges facing the transplant community today is the lack
of good quality organs. Facing the prospect of a widening deficit between organ
supply and demand in the next ten years, there becomes an urgent need to
develop novel strategies to protect, resuscitate and repair donor organs. And thus
improve organ quality and the outcomes for transplant recipients. Addressing this
quality deficit is particularly important for organs obtained from extended criteria
donors (older brain dead donors with additional co-morbidities) and donors after
circulatory arrest.
The QUOD is a national NHSBT funded programme led by Professor Rutger J Ploeg,
Professor of Transplant Biology at the University of Oxford and Consultant Surgeon at
the Oxford University Hospital OUH NHS Trust. The key aims of this initiative are:
• to increase the number and quality of organs procured from brain dead donors and
donors after cardiac death, by optimising donor management, resuscitating and
preserving high-risk donor organs
• to make previously unusable organs transplantable and increase the ‘donor pool’
• to identify pathways of injury and apply targeted interventions to repair donor organ
injury
• to translate validated experimental methods and technologies into clinical use and
best practice protocols, and
• to identify biomarkers and functional parameters that predict outcome following
transplantation.
RECENT PUBLICATIONS
Moers C, Pirenne J, Paul A, Ploeg RJ.
Machine perfusion or cold storage in
deceased-donor kidney transplantation.
N Engl J Med. 2012, 366(8):770-1
Damman J, Seelen MA, Moers C, et
al. Systemic Complement Activation
in Deceased Donors Is Associated
With Acute Rejection After Renal
Transplantation in the Recipient.
Transplantation, 2011, 92 (2): 163-169
Jochmans I, Moers C, Smits JM, et al.
The prognostic value of renal resistance
during hypothermic machine perfusion
of deceased donor kidneys. Am J
Transplant. 2011, 11(10), 2214-20
Nijboer WN, Schuurs TA, Damman J, et
al. Kidney injury molecule-1 is an early
noninvasive indicator for donor brain
death-induced injury prior to kidney
transplantation. Am J Transplant. 2009,
9 (8):1752-9
We aim to achieve these key goals by forming a national Consortium consisting of
experts from the intensive care and transplant units from across the United Kingdom in
conjunction with NHSBT. We will collect information from appropriately consented donors,
including biological specimens, analyse samples and correlate this to outcomes following
transplantation. By doing this we will identify novel markers that can be used to predict
the function and outcomes of transplantation, but also improve our understanding of
the underlying pathways of donor organ injury. The Consortium will create a platform for
trialling new therapeutic strategies aimed at resuscitating and repairing damaged donor
organs to improve immediate function and increase long-term survival.
Contribution to patient and donor care or service development
By targeting organ injury and improving organ quality we will increase the absolute number
of life saving organs available for transplantation. In addition, better short and long-term
outcomes after transplantation will improve organ and patient survival, reduce postoperative complications and the need for re-transplantation.
Research and Development Triennial Report 2009-12
45
Theme
1
6
Organ donation and transplantation
Impact of donor factors on transplant outcome
Professor Dave Collett
[email protected]
CONTRIBUTING TEAM MEMBERS: Rachel Johnson, James Neuberger, Dominic Summers, Rhiannon Taylor
and Helen Thomas
The shortage of donor organs for transplantation means that surgeons need to
consider using organs that are higher risk or sub optimal. Recent work in this area
has looked at using kidneys from ‘donation after circulatory-death’ (DCD) donors,
and lungs from donors with a positive smoking history.
RECENT PUBLICATIONS
Ausania F, White SA, Pocock P,
Manas DM. Kidney Damage During
Organ Recovery in Donation After
Circulatory Death Donors: Data From
UK National Transplant Database.
American Journal of Transplantation
2012; 12: 932-936
Kidneys from DCD donors
Until the last ten years, the vast majority of kidneys for transplantation in the UK came
from heart-beating, brain-dead, donors. However, brain-death is quite a rare event in the
UK (1,176 cases in 2011), and so even if all these donors were fit enough to donate and all
their families consented to donation, there would still be too few organs available for the
7,000 patients waiting. One solution to this shortage has been the use of DCD donors,
in which death of the donor is declared after cardio-respiratory arrest, following the
controlled withdrawal of treatment in patients who have suffered catastrophic injuries.
Bonser RS, Taylor R, Collett D,
Thomas HL, Dark JH and Neuberger J.
The smoking donor – benefit
or hazard to the potential lung
recipient? Lancet 2012; Early Online
Publication, 29 May 2012
There have been concerns about using organs from this source, as the organs will
undergo a prolonged period with an inadequate blood supply, potentially causing them
permanent damage. We have been able to show, for the first time, that kidneys from
DCD donors work as well as kidneys from brain-dead donors in the longer term and
that kidneys from older DCD donors are no worse than kidneys from older brain-dead
donors. In addition, we have found that DCD donor kidneys are more susceptible to
the damage incurred during cold storage of the organ prior to transplantation. This has
implications for the allocation of such kidneys, in that long ischemic times associated
with transporting the organ from the retrieval to implantation centre must be avoided.
Summers DM, Johnson RJ,
Allen J, Fuggle SV, Collett D,
Watson CJ, Bradley JA. Analysis of
factors that determine outcome
following transplantation with
kidneys donated after cardiac death
in the UK. Lancet 2010; 376: 13031311
Summers DM, Counter C,
Johnson RJ, Murphy PG,
Neuberger JM, Bradley JA. Is the
Increase in DCD Organ Donors in
the United Kingdom Contributing
to a Decline in DBD Donors?
Transplantation 2010; 90:1506-1510
Lungs from smoking donors
The shortage of lungs for transplantation means that around 40% of lungs come from
donors with positive smoking history (PSH). However, recipients of such lungs have a
significantly worse survival after transplantation (see chart). In view of this, an important
question facing the patient is whether to accept an offer of lungs from a PSH donor or wait
for lungs from a non-smoker. Using a novel statistical analysis, we showed that patients
Articles
receiving lungs from donors with positive smoking histories had a longer survival time
following registration than did those who remained on the waiting list. The continued use of PSH donor lungs is therefore warranted.
Contribution to patient and donor care or service development
46
had slightly higher body-mass indices than those who
received lungs from donors with negative smoking
histories; other characteristics were similar between
groups (table 2). Use rates of lungs from donors with
positive smoking histories did not differ significantly
between transplantation centres (table 2).
Unadjusted Kaplan-Meier estimates of post-transplantation survival showed inferior survival by donor
history of smoking (figure 2A); median post-transplantation survival time was 4·9 years (95% CI 4·4–5·5) for
transplants from donors with positive smoking histories
Research
Development
Triennial
Reportfrom
2009-12
and and
6·5 years
(5·9–7·2) for
transplants
donors with
negative smoking histories. Survival was already inferior
30 and 90 days after transplantation (table 2). The
Survival (%)
Our findings have reassured transplant clinicians and recipients that organs from DCD donors can safely be used, increasing the
number of kidneys available for patients on the waiting list. We have also been able to tailor the national kidney allocation policy
so that
thepositive
storagesmoking
time forhistories
these kidneys
is minimised
with
had similar
clinical and
A
donor–recipient
matching
characteristics
donors with
to optimise
transplant
outcome.
In regard toto lung
100
NSH donor
negative smoking
histories,
exception
small
transplantation,
although
lungswith
fromthe
PSH
donorsofare
PSH donor
but
significant
differences
in
cytomegalovirus
matching
associated with a poorer outcome, an organ selection
80
and cause
death such
(tabledonors
1). We noted
no significant
policystatus
that uses
lungsoffrom
improves
overall
differences in donor’s age, oxygenation, size matching,
survival
of patients registered for lung transplantation, and
60
and ischaemic time (table 1). Recipients of lungs from
shoulddonors
be continued.
with positive smoking histories were older and
40
1 year
NSH donor 81·3 (78·1–84·1)
PSH donor 74·6 (70·4–78·3)
Log-rank p value 0·006
20
0
Number at risk
NSH donor
PSH donor
3 years
67·2 (63·2–70·8)
55·7 (50·7–60·4)
0·0002
5 years
57·4 (53·0–61·6)
49·2 (43·9–54·3)
0·002
0
1
2
3
4
5
6
709
502
470
301
370
216
294
169
239
144
189
108
137
88
B
100
80
Theme
Organ donation and transplantation 1
6
Allocation of organs for transplantation
Rachel Johnson
[email protected]
CONTRIBUTING TEAM MEMBERS: Joanne Allen, Kerri Barber, Dave Collett, Alex Hudson, David Manlove,
Gregg O’Malley and John O’Neil
Living donor kidney transplantation for incompatible
donor-recipient pairs
RECENT PUBLICATIONS
The shortage of organs for transplantation means that organ allocation schemes are
needed to ensure that UK patients have equity of access to organs from deceased
donors and that opportunities for exchanging living donor kidneys are maximised.
Recent work has focused on novel arrangements for allocating kidneys, the allocation
of pancreases and islets, and approaches to the allocation of livers.
Johnson RJ, Allen JE, Fuggle SV,
Bradley JA, Rudge CJ. Early Experience
of Paired Living Kidney Donation in the
United Kingdom. Transplantation 2008;
86:1672-1677
Preparing a DCD donor kidney for transplantation
A potential living kidney donor
and his/her intended recipient are
biologically incompatible in about
one-third of potential donorrecipient pairs. In this situation, a
number of transplant options may
be possible, including kidneypaired exchange (KPE), whereby
incompatible pairs exchange
donor kidneys to circumvent
incompatibility, waiting for a
deceased donor transplant, or
proceeding with an incompatible
transplant with appropriate
treatment.
A study with the University of Glasgow examines possibilities for extending living donor
kidney transplantation through KPE, and to identify optimal strategies for transplantation
for immunologically incompatible donor-recipient pairs, based on the nature of the
incompatibility between donor and recipient. This enables a more effective and informed
pathway to transplantation in terms of waiting time, transplant outcome and cost.
Johnson RJ, Fuggle SV, Mumford L,
Bradley JA, Forsythe JLR, Rudge CJ.
A new UK 2006 national kidney
allocation scheme for deceased heart
beating donor kidneys. Transplantation
2010; 89: 387-394
Watson CJE, Johnson RJ, Birch R,
Collett D, Bradley JA. A simplified
donor risk index for predicting
outcome after deceased donor kidney
transplantation. Transplantation 2012;
93: 314-318
Barber K, Pioli S, Blackwell J,
Collett D, Neuberger J, Gimson A.
Elective liver transplant list mortality:
development of a United Kingdom
end-stage liver disease score.
Transplantation 2011; 92(4):469-76
Allocation of pancreas and islets
A new National Pancreas Allocation Scheme was introduced on 1 December 2010. Patients are prioritised according to a points
system based on a range of clinical factors. A computer program calculates a score for every potentially suitable patient on the
national active transplant list and the pancreas is allocated preferentially to the patient with the most points. Pancreases from
donors after brain death and donors after circulatory death are allocated through this scheme. Patients listed for a vascularised
pancreas or islet transplant are prioritised through one combined national transplant list.
The new scheme aims to reduce the incidence of long waiting patients and to improve equity in access to transplant irrespective
of where in the UK each patient resides, without adversely affecting graft survival.
Allocation of livers
At present, livers for elective transplantation are allocated to transplant centres, which then decide which patient receives the
organ. A number of alternative schemes have been developed that prioritise patients according to need (liver offered to sickest
patient), utility (liver offered to maximise life years of graft) and benefit (liver offered to patient predicted to gain most life years
following transplant). Although there are limitations in the data available, our results suggest that an allocation system based on
need or benefit is comparable to the current scheme.
Contribution to patient and donor care or service development
Up to the end of March 2012, about 150 patients had been transplanted through the kidney paired exchange programme. The
introduction of a national pancreas allocation scheme has allowed more patients to benefit from islet transplantation without
having an adverse effect on the pancreas transplant programme.
Research and Development Triennial Report 2009-12
47
Theme
1
6
Organ donation and transplantation
MALIGNANCY FOLLOWING TRANSPLANTATION
Cancer in transplant recipients
types, so that for example, the SIRs tend to be
Figure 3: Examples of malignancies in transplant recipients.
higher for lung transplant recipients. The incidence of some cancers, and particularly NMSC,
Professor James Neuberger
may be underestimated because of incomplete
[email protected]
reporting to the cancer
registries. These results
suggest that there is a case for regularly screening for NMSC, lip and anal cancer, but no need
for additional surveillance of breast or cervical
CONTRIBUTING TEAM MEMBERS: Joanne Allen, Kerri Barber, Dave Collett, Alex Hudson, David Manlove,
cancer1.
Gregg O’Malley and John O’Neil
Different cancers exhibit different SIRs over
time1. To illustrate this, Figures 1 and 2 show
Thepatterns
occurrence
a recognised
adverse event following organ
the
in of
themalignancy
SIRs overistime
following
transplantation.for
OurNMSC
first national
studyrespectof malignancy linked data from British
transplantation
and PTLD,
transplant
recipients
to information
on cancer
ively.
The SIR
for NMSC
increases steadily
overregistrations obtained from the
regional
cancer registries
in England
and the national registries of Scotland
time
in recipients
of a kidney
or cardiothoracic
organ,
but for
recipients,
the SIR has
and Wales.
Theliver
linkage
allowed calculation
of athe time to first registration of a
much
lower following
value andsolid
reaches
maximum
malignancy
organ its
transplantation.
within two years. In contrast, the SIR for PTLD
non-melanoma
skin cancer
inOverall,
kidney,incidence
liver andoflung
recipients peaks
at one(NMSC) was found to be 14 times that
of the
general
population, while
thetwo
overall
incidence
rate for all other cancers is just
year
after
transplantation
and at
years
for
heart
transplant
recipients.
Inpopulation.
terms of absolute
over twice
that of
the general
Many forms of cancer have a much higher
risk,
malignancy
incidence
in kidney
recipients
incidence
in transplant
recipients
than the
general population, especially cancer of the
islip,
low
in younger lymphoproliferative
recipient age groups,
and
post-transplant
disease,
cancer of the oral cavity and anal cancer.
increases
age; it is also
males
There arewith
also differences
in thegreater
patternfor
of incidence
between the different transplant
than
Examples
of incidence
post-transplant
types,females.
so that, for
example, the
of NMSC is lower for liver transplant recipients
malignancies
are shown in Figure 3.
(see chart below).
Standardised incidence ratio
25
Kidney
Liver
Heart
Lung
20
15
10
5
0
0
3
6
9
Years since transplantation
12
15
Figure 1: Changes in the SIR over time since
transplantation for NMSC in recipients of different organs.
The increased risk of development
of(1)a An
malignancy
inshowing
a transplanted
MR of the brain
a glioblastoma.
patient is mainly due to the use of
immunosuppression, but there is
also the possibility of transmission
of a cancer from a donor. Outcome
for the recipient is poor when such
transmission occurs, with a high
chance of graft loss and death.
Because of this, donors with a
known history of malignancy
are not generally considered for
transplantation.
RECENT PUBLICATIONS
Collett D, Mumford L, Banner NR,
Neuberger J, Watson CJE. Comparison
of the incidence of malignancy
in recipients of different types of
organ: a UK registry audit. American
Journal of Transplantation 2010;10:
1889–1896
Watson CJE, Roberts R, Wright KA,
Greenberg DA, Rous BA, Brown CH,
Counter C, Collett D, Bradley JA.
How safe is it to transplant organs
from deceased donors with primary
intracranial malignancy? An analysis of
UK registry data. American Journal of
Transplantation 2010; 10: 1437–44
Warrens AN, Birch R, Collett D,
Daraktchiev M, Dark JH, Galea G,
Gronow K, Neuberger J, Hilton D,
Whittle IR, Watson CJE. Advising
potential recipients on the use of
organs from donors with primary
central nervous system tumours.
Transplantation 2012; 93(4):
348-353
Estimating risk of transmission
60
Kidney
Standardised incidence ratio
While national and international guidelines
Liver have been developed, these usually have a very weak evidence base and our
50 data suggest that, for brain cancers
own
at least, exclusion of some donors will actually increase patient mortality because
Heart
Lung
of the
high
risk
of
death
awaiting
a
transplant.
To estimate the risk of transmission, recipients with donor-transmitted cancer
40
were identified from the UK Transplant Registry and a database search at transplant centres.
30
(3) A with
colon with
multiple ulcerated
areaswith
of PTLD
in a patient 8 years
after aoccurred
kidney transplant.
We found 15 out of 30,765 transplant recipients (0.05%)
transmitted
cancer
transmission
having
from
13 20
out of 14,986 donors (0.09%). Donors aged 45 years or more were nine times more likely to result in cancer transmission
theproportion
type of immunosuppression,
but this
than donors aged less than 45 years. This is importantand
as the
of older donors is increasing
in the UK, and now
10
be the
subject of in
further
work.
more
than half of all organ donors are aged 50 years will
or more.
Furthermore,
none of
these cases was the presence of
0
tumour
known before transplantation.
0
3
6
9
12
15
Years since transplantation
TRANSMISSION
Contribution to
patient and donor care or service
developmentOF MALIGNANCY
Figure 2: Changes in the SIR over time since
transplantation
PTLD in
recipientshelps
of different
organs. patient
The
development
a malignancy
a transOur study onfor
cancer
incidence
to identify
groups
and cancer of
types
which shouldinform
part
of a cancer
planted
patient ispatients
mainlyondue
use of
surveillance programme, and provide data for counselling
pre-transplant
the to
riskthe
of malignancy.
The precise risk
immunosuppression,
butdata
there
is also
theis a rare event.
Based
on limited
there
no to
evidence
of transmission
of adata,
cancer
fromwas
donor
recipient is unknown,
but the available
suggest
that this
of an association between cancer incidence
possibility of transmission of a cancer from a
This small risk of tumour transmission needs to be balanced against the likely mortality for potential recipients who remain
on the transplant list. Patients can now be counselled more accurately on the benefits as well as the risks of transplantation.
4
48
Research and Development Triennial Report 2009-12
British Journal of Transplantation
Theme
Stem cells and immunotherapies 1
7
Immunogenetic markers in transfusion and
transplantation
Dr Cristina Navarrete and Dr John Girdlestone
[email protected], [email protected],
[email protected], [email protected]
Dr Chistina Navarrete
Dr John Girdlestone
CONTRIBUTING TEAM MEMBERS: Sandra Cardoso, Winnie Chong, Lisa Creary, Delordson
Kallon and Alina Lemnrau
Appropriate matching between donor and recipient is critical for the safe
and effective transfusion of blood, and for the transplantation of organs or
haematopoietic stem cells. We are dedicated to developing new and more
efficient strategies and techniques for analysing the genetic markers that
contribute to optimal matching and successful patient clinical outcomes including
alleles of the HLA, HPA and HNA antigenic systems. In addition, we are also
investigating the role of HLA ligand/Killer Immunoglobulin Receptor (KIR) pairing
in the outcome of UCB stem cell transplants.
The NHS Cord Blood Bank has now issued nearly 400 units for transplantation
worldwide. We are performing a retrospective analysis of donor and recipient
HLA alleles to determine if clinical outcomes are improved by high resolution (HR)
matching for class I (HLA-A, -B, -C) and class II (HLA-DRB1, -DQB1) loci compared to
the current method of matching at low/mid resolution at class I loci and HR at -DRB1.
HLA ligand and KIR combinations in these transplants are also being determined to
see if mis-matching conveys a benefit in cord blood transplants for leukaemia patients
and to develop methods for more efficient, cost-effective typing of HLA, HPA and
HNA alleles.
While matching of individual HLA alleles is known to be important for clinical
transplantation outcomes, there is increasing evidence that genes linked on the
same chromosomal region (a haplotype) can also have an effect. To capture this
information, we have developed a method using HLA TagSNPs to identify the three
most common Caucasoid haplotypes.
RECENT PUBLICATIONS
Brown CJ, Navarrete CV. The clinical
relevance of the HLA system in blood
transfusion. Vox Sang 2011; 101:
93-105
Hollenbach JA, et al. Worldwide
variation in the KIR loci and further
evidence for the co-evolution of KIR
and HLA. Tissue Antigens. 2010;
76:9-17
Chong W, et al. Detection of human
platelet antigen-1a alloantibodies in
cases of fetomaternal alloimmune
thrombocytopenia using recombinant
β3 integrin fragments coupled
to fluorescently labelled beads.
Transfusion. 2011;51:1261-70
Alina Alina Lemnrau Identification
of Human Leukocyt Antigens (HLA)
haplotypes using tagSNPS. PhD
Thesis, UCL. 2012
Patients requiring repeated transfusion of platelets for clotting disorders can develop
antibodies against foreign HLA as well as HPA alleles, making it difficult to find a
compatible products when needed. We are also developing new approaches using
molecular and bioinformatic tools to define HLA epitopes (Eplets) for the provision of
compatible products for these patients.
Antibodies to HPA can cause serious complications during pregnancy in addition to
poor responses in patients requiring platelet transfusions, and in collaboration with
other NHSBT R&D laboratories we have developed a multiplexed method employing
recombinant proteins for the screening of common HPA antibodies.
Contribution to patient and donor care or service development
To identify the most appropriate level of matching for selecting donors that can
provide the best clinical outcome of transplantation and transfusion, and to develop
methods for more efficient, cost-effective typing of HLA, HPA and HNA alleles.
The identification of more cost effective and
clinically relevant diagnostic tests for HLA and other
genetic markers such as SNPs are being investigated
for their roles in transplantation and transfusion.
The identification of HLA tagSNPs that identify
common HLA haplotypes led to the development of
a multiplex assay using a platform that is in standard
use in the Histocompatibility and Immunogenetics
(H&I). This approach has now been transferred to
the Colindale-based H&I Service Development group
for translation to the NHSBT H&I Service.
Prediction of permissible HLA mismatches.
Prediction of permissible HLA mis-matches. The original MatchMaker program of
Duqesnoy used contiguous triplets of amino acids to compare HLA alleles, but the newest
version looks at spatially adjacent residues that more closely represent antibody-binding sites.
Adapted from: Kostyu et al. Human Immunology 57, 1-18, 1997
The original MatchMaker program of
Duqesnoy used contiguous triplets of
amino acids to compare HLA alleles,
but the newest version looks at
spatially adjacent residues that more
closely represent antibody-binding
sites.
Adapted from: Kostyu et al. Human
Immunology 57, 1-18, 1997
In order to provide the best possible
matching between donors and patients,
we develop DNA and protein assays
for
mult-allelic genetic systems.
In order to provide the best
possible matching between
donors and patients, we
develop DNA and protein
assays for multi-allelic
genetic systems.
Research and Development Triennial Report 2009-12
49
Theme
1
7
Stem cells and immunotherapies
Immunoregulatory properties of cells derived
from umbilical cord and cord blood
Dr Cristina Navarrete and Dr John Girdlestone
[email protected], [email protected],
[email protected], [email protected]
Dr Chistina Navarrete
Dr John Girdlestone
CONTRIBUTING TEAM MEMBERS: Antony Cutler, Sima Hirani and Meera Raymond
Umbilical cord blood (UCB) is increasingly being used as an alternative to bone
marrow or mobilised peripheral blood as a source of stem cells for reconstitution
of the haematopoietic and immune systems of patients receiving treatment for
leukaemias or non-malignant diseases. The use of UCB is associated with lower
rates of a life-threatening complication, acute Graft versus Host Disease (aGvHD),
compared to that seen when stem cells from adult donors are used. The main
aims of our group are to understand the unique characteristics of immune
cells in UCB that could influence this outcome, and also to determine how the
immunosuppressive properties of Mesenchymal Stromal Cells (MSC) derived from
the umbilical cord (UC) itself may be exploited to treat aGvHD.
It has been shown that although the proportion of regulatory T-cells (T-regs) are
similar in adult and cord blood CD4 compartments, there is a very low level of
activated, CD25+ T-effector cells in UCB, such that the ratio of T-regs to T-effector
is much higher in UCB. The relative numbers and/or activity of T regulatory cells
(T-reg) may also contribute to the reduced GVHD seen following UCB transplantation.
On the other hand MSC have been used as feeder layers for the expansion of CB
haematopoietic stem cells, and we and other groups have shown that co-culture of
CD4+ lymphocytes with MSC can not only maintain but also induce, T-reg numbers
during lymphocyte expansion.
If patients suffering from aGvHD fail to respond to steroids then their treatment
options are limited. A number of groups have reported favourable responses upon
treatment with MSC, and clinical trials are now underway around the world to
determine their efficacy. The majority of these trials are testing MSC derived from
bone marrow, but we and other researchers have shown that the UC can serve as a
less invasive, more convenient source of MSC. We have shown that the UC-derived
MSC are capable of inhibiting immune reactions, and demonstrated that monocytes
play a crucial role in mediating their suppression of T-cell proliferation.
The naive state of immune cells in UCB is thought to contribute to their reduced
tendency to cause GvHD. However, immune regeneration is reported to be slower
with UCB, which leaves patients at greater risk of life-threatening infections. Clinical
trials on the use of UCB are now underway in the UK and we are collaborating with
transplant centres to study the immune reconstitution of their recipients in order to
understand how best to monitor and understand this process.
Haematopoietic stem cell transplantation can be a life-saving treatment, but
transplant-related mortality remains a serious problem. Through increased
understanding of the parameters that influence the development of aGVHD, stem
cell engraftment and immune reconstitution, we have the potential to identify the
most appropriate stem cell grafts for achieving successful transplants with minimal
side effects. Advances in this area will increase patient survival and quality of life, and
reduce demands on healthcare resources.
50
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Girdlestone J, et al. Efficient
expansion of mesenchymal stromal
cells from umbilical cord under
low serum conditions. Cytotherapy
2009;11:738-48
Cutler AJ, et al. Umbilical cord
derived mesenchymal stromal
cells modulate monocyte function
to suppress T-cell proliferation.
J Immunol 2010; 185: 6617-6623
Gluckman E, et al. Family-directed
umbilical cord blood banking.
Haematologica. 2011; 96:1700-1707
Hirani S. The characterisation
of human umbilical cord blood
regulatory T-cell subsets. PhD Thesis,
UCL Feb 2012
Theme
Stem cells and immunotherapies 1
7
Immune reconstitution in recipients of UCB is monitored using flow cytometry.
Immune
reconstitution
in recipients
of UCB
usingNK
flow
A patient
sample
(left) contains
monocytes
(red),isTmonitored
(d. blue) and
(I. blue) cells,
cytometry.
A patient
sample
(left) contains
but very
few B Cells
(green)
compared
to UCBmonocytes
(right). (red),
T (d. blue) and NK (l. blue) cells, but very few B Cells (green) compared
to UCB (right).
Monocytes are required for inhibition of T-Cell proliferation by MSC.
Proliferation of T-cells in response to anti-CD3/CD28 (black) is inhibited by MSC (red), but
this suppression
is blocked
upon the
of monocytes
Monocytes
are required
forremoval
inhibition
of T Cell (yellow) but not B cells (blue).
proliferation by MSC. Proliferation of T cells in
response to anti-CD3/CD28 (black) is inhibited by
MSC (red), but this suppression is blocked upon
the removal of monocytes (yellow) but not B cells (blue).
Expanded CB T-reg are immunosuppressive.
Freshly isolated (day 1) CB T-reg (CD25+) are weakly suppressive compared to those
fromExpanded
adults (AB),CB
butTreg
become
equally potent upon expansion
vitro. (day1)
are immunosuppressive.
Freshlyinisolated
CB Treg (CD25+) are weakly suppressive compared to those from
adults (AB), but become equally potent upon expansion in vitro.
Research and Development Triennial Report 2009-12
51
Theme
1
7
Stem cells and immunotherapies
Improving cord blood transplant outcomes by
understanding the bone marrow niche
Professor Suzanne Watt
[email protected]
CONTRIBUTING TEAM MEMBERS: B Zhou, Y Zhang, CT Yang, G Tsaknakis, K Pilkington, E Pepperell,
E Martin-Rendon, S Lee, C Khoo, S Hale, A French, R Forsey, K Coldwell, C-H Chang, L Carpenter and S Buglass
Umbilical cord blood is an important source of blood stem cells for regenerating
a normal blood supply following transplantation for diseases such as acute
leukaemias. Key challenges include limited stem cell numbers, repair of the
damaged bone marrow niche following chemotherapy/radiotherapy and delayed
blood cell regeneration within the bone marrow niche when compared to other
sources of blood stem cells. This can lead to graft failure and/or to increased
susceptibility to bleeding or infections post transplant and hence resultant
morbidity and mortality.
Enhancing cord blood stem/progenitor cell efficacy before grafting can prevent
engraftment delays and improve transplant outcomes, but strategies used can
be prohibitively expensive to health providers or may still be in development, and
therefore may not be adopted immediately by transplanters.
Our particular objectives are to improve the therapeutic use of cord blood stem/
progenitor cells by first understanding how their homing to bone marrow and their
self-renewal and development into blood cells are regulated by the bone marrow
microenvironmental niche, and then by developing cheaper and affordable methods
for the enrichment and expansion of cord blood stem cells which will efficiently ‘take’
after transplantation.
In achieving these goals, one of our key successes has been to develop a novel in
vitro time-lapse method for assessing trafficking of individual human cord blood
stem/progenitor cells towards the key bone marrow attractant, CXCL12. This assay
distinguishes between directed homing (chemotaxis) and random movement
(chemokinesis) of cord blood stem/progenitor cells, thereby allowing refinement of
conditions for pre-transplant manipulation of these stem/progenitor cells so that their
homing abilities can be assessed and optimised prior to transplant.
This assay represents an important advance when analysing rare stem/progenitor
cell populations from or for use in patients. We are also using different technologies,
including cell reprogramming, scaffolds and bioreactors combined with novel factors
and niche elements, and non coding RNAs or small molecules, to expand cord
blood stem/progenitor cell numbers, to repair the damaged niche or to modulate
interactions of cells within the niche.
Interestingly, we have identified candidate co-receptors for the CXCL12 receptor,
CXCR4, which may influence stem cell homing, reprogrammed CD34+ cord blood
and other cells which can generate haemopoietic and vascular lineages, and we
were the first to demonstrate that a clonal bone marrow MSC line could promote
both haemopoiesis and vasculogenesis. Such MSC subsets may thus have a dual
regulatory role in promoting haematological recovery in the transplant setting. These
developments will contribute substantially to achieving the desired outcomes.
52
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Zhou B*, Tsaknakis G*, Coldwell KE,
Khoo CP, Roubelakis MG, Chang CH,
Pepperell E, Watt SM. A novel
function for the haemopoietic
supportive murine bone marrow
MS-5 mesenchymal stromal cell line
in promoting human vasculogenesis
and angiogenesis. Brit J Haematol.
2012; 157(3): 299-311
(*joint 1st authors)
Pepperell EE, Watt SM. CXCL12induced trafficking of individual
hematopoietic CD133+ stem/
progenitor cells in vitro. 2012
Coldwell KE, Lee SJ, Kean J, Khoo CP,
Tsaknakis G, Smythe J, Watt SM.
Effects of obstetric factors and
storage temperatures on the yield of
endothelial colony forming cells from
umbilical cord blood. Angiogenesis.
2011;14(3):381-392
Carpenter L, Malladi R, Yang CT,
French A, Pilkington KJ, Forsey RW,
Sloane-Stanley J, Silk KM, Davies TJ,
Fairchild PJ, Enver T, Watt SM. Human
induced pluripotent stem cells are
capable of B-cell lymphopoiesis.
Blood. 2011;117(15): 4008-4011
Stem cells and immunotherapies Theme
1
7
Contribution to patient and donor care or service development
NHSBT has taken a leading role in cord blood stem cell provision by establishing the first public NHS Cord Blood
Bank in the UK for clinical transplantation. Our research contributes to patient care and service development through
improvements in cord blood stem cell treatments, allowing these to be made more widely available to patients in the
NHS and worldwide and complementing the NHSBT strategy for cord blood.
The clonal mesenchymal stem/stromal cell
line, MS-5, produces specific angiocrine
factors (middle array), which support blood
vessel formation (top image) and mediate
human blood stem cell homing to the bone
marrow niche.
Research and Development Triennial Report 2009-12
53
Theme
1
7
Stem cells and immunotherapies
Stem cell therapies for heart disease
Dr Enca Martin-Rendon and Professor Suzanne M Watt
[email protected]
[email protected]
CONTRIBUTING TEAM MEMBERS: David M Clifford, Dominic Sweeney, George N Thomas,
Dr Enca Martin-Rendon
Professor Suzanne M Watt
Huajun Zhang
Heart disease is the major cause of death in the world and the number of patients
suffering from heart failure, following a heart attack, is steadily increasing. Stem
cell therapies are a promising novel treatment that may reduce the number
of patients who later die or suffer from heart failure. However, we still do not
understand fully how stem cells work and how the disease affects them.
Ischaemic heart disease is characterised by the poor supply of blood from the
coronary arteries to the myocardium, with the consequence of damaging the heart
and causing heart failure in the long-term. There is an unmet clinical need to find
alternative therapies as the number of patients requiring heart transplants increases.
In collaboration with the Systematic Review Group at NHSBT Oxford, we have
evaluated clinical evidence from randomised trials where bone marrow stem cells are
administered to patients following a heart attack. We have demonstrated that stem
cell treatment significantly improves contractility and heart function and significantly
reduces the size of the infarct long-term. Factors such as stem cell dose, route of
delivery and heart function at baseline could influence the outcome of the therapy.
In addition, our research aims to improve the formation of blood vessels in the
damaged heart following a heart attack. Our clinical collaborators are Professors
David P. Taggart (John Radcliffe Hospital, Oxford) and Anthony Mathur (The London
Chest Hospital). We are investigating how stem cells from tissues like the bone
marrow can help support the formation of new blood vessels in the damaged heart.
We are particularly interested in a population of mesenchymal stem cells that can be
identified by the cell surface markers CD90 and CD105. We have also established a
protocol by which we can readily expand these cells from the patients’ own heart
tissue. The number and function of the stem cells derived from the heart can be
affected by cardiovascular risk factors, such as diabetes and hypertension, and by
advanced state of disease. This work will improve our understanding of the underlying
mechanisms that regulate stem cell function.
Contribution to patient and donor care or service development
Stem cell therapy for cardiac repair is not a product that NHSBT currently offers to
patients. Although the Stem Cell and Immunotherapies (SCI) service laboratory in
Bristol has contributed to clinical grade CD133+ cell selection for collaborative clinical
trials (TRANSACT I and II) with the Bristol Heart Institute. Stem cells will hopefully
provide personalised treatments and biologics for diseases other than haematological
disorders. Our aim is to make stem cell-related therapies a product or service that
NHSBT could be offering to patients suffering from cardiovascular diseases in the
future.
Stem cell therapy for ischaemic heart disease. Bone marrow: (1) and
heart-derived (2) stem cells are being investigated as treatment for patients
suffering from ischaemic heart disease. Autologous stem cell transplantation
may have the potential to reduce the number of patients who die or develop
heart failure following a heart attack.
54
Research and Development Triennial Report 2009-12
RECENT PUBLICATIONS
Chan HL, Meher Homji Z,
Gomes RSM, Sweeney D,
Thomas GN, Tan JJ, Zhang H,
Perbellini F, Stuckey DJ, Watt SM,
Taggart DP, Clarke K,
Martin-Rendon E and Carr CA.
Human cardiosphere-derived cells
from patients with chronic ischaemic
heart disease can be routinely
expanded from atrial but not
epicardial ventricular biopsies.
Journal of Cardiovascular Translational
Medicine 2012 July3.
Clifford DM, Fisher SA, Brunskill SJ,
Doree C, Mathur A, Clarke M,
Watt SM, Martin-Rendon E.
‘Long-term effect of autologous
bone marrow stem cell treatment in
acute myocardial infarction: Factors
that may influence outcome’ 2012a.
PLoS ONE; 7(5):e37373.
Clifford D, Brunskill SJ, Fisher S,
Doree C, Mathur A, Watt SM,
Martin-Rendon E. (2012b) Stem
Cell Treatment for Acute myocardial
Infarction. Cochrane Database Syst.
Rev, Feb 15, 2012; 2:CD006536.
4. Brunskill SJ, Hyde CJ, Doree CJ,
Watt SM and Martin-Rendon E
(2009) Route of delivery and baseline
left ventricular ejection fraction, key
factors of bone marrow-derived cell
therapy for ischaemic heart disease.
Eur. J. Heart Fail. Sep; 11(9):887-96.
Theme
Stem cells and immunotherapies 1
7
Regulatory T-cell therapy for graft
verse host disease following human
stem cell transplantation
Dr Wei Zhang, Professor Suzanne Watt and
Professor David Roberts
Dr Wei Zhang
Professor Suzanne M Watt
Professor David Roberts
[email protected], [email protected] and [email protected]
CONTRIBUTING TEAM MEMBERS: Robert Danby, Andy Peniket, Emma Frith, Sindu Nair, Jon Smythe, Sophie Clark and Sylvia Benjamin
Graft versus Host Disease (GvHD) remains a major complication following human
stem cell transplantation (HSCT). Regulatory T-cells (T-regs) are a naturally-occurring
subpopulation of T-cells that have been shown to selectively reduce GvHD and while
allowing a Graft versus Leukaemia effect (GvL) in both mouse SCT models and in
limited phase I clinical HSCT trials. However, the precise mechanisms of T-reg action
remain elusive. We are developing the application of T-regs as a cellular therapy in
different HSCT settings to reduce GvHD and also exploring the mechanism of action
of T-regs in GvHD.
RECENT PUBLICATIONS
Rayment R, Kooij TW, Zhang W, Siebold
C, Murphy MF, Allen D, Willcox N,
Roberts DJ. Evidence for the specificity
for platelet HPA-1a alloepitope and
the presenting HLA-DR52a of diverse
antigen-specific helper T-cell clones from
alloimmunised mothers. J Immunol.
2009 Jul 1;183(1):677-86
With Robert Danby and Andy Peniket (Department of Haematology, John Radcliffe
Hospital, Oxford) we have measured T-regs in both the graft and in the peripheral blood
of 110 patients treated at Oxford Cancer Centre over the last three years. We found a
high number of T-regs in the graft correlated with improved engraftment and improved
overall survival. Our data therefore support T-regs as a therapeutic intervention to
improve the outcome in Haematopoietic Stem Cell Transplantation (HSCT).
Nadig SN, Wieckiewicz J, Wu DC,
Warnecke G, Zhang W, Luo S, Schiopu
A, Taggart DP, Wood KJ. In vivo
prevention of transplant arteriosclerosis
by ex vivo-expanded human regulatory
T-cells. Nat Med. 2010 Jul;16(7):809-13
We have validated the standard operating procedure for the Good Manufacturing
Practice (GMP) isolation of T-regs in close collaboration with Stem Cell Service and
Specialist Therapeutic Services in Oxford. We are proposing a phase I/II clinical study
with Prof Vanderson Rocha and Andy Peniket (Department of Haematology, John
Radcliffe Hospital, Oxford) in patients with reduced conditioning for HSCT.
Group webpage and websites
http://www.ndcls.ox.ac.uk/
ResGroups.php?GID=3
We have tested conditions for expansion of T-regs in an attempt to increase their
number and function. We found a subset of mature T-regs expressing the highest levels of FoxP3 are highly sensitive to
Fas-FasL induced cell death. The cell populations after expansion are therefore depleted of functional T-regs but enriched for
cytokine producing conventional T-cells. In vitro expanded T-regs have also been found to be short lived after transfusion into
patients. Our data therefore caution the use of T-regs expanded in the laboratory.
% cells CD25+FOXP3+
% cells CD25+FOXP3+
% cells CD25+FOXP3+
Various cell surface molecules and soluble factors have been implicated in the function of T-regs. In response to TCR
stimulation, FoxP3 – a crucial transcription factor for T-regs – was up-regulated and
modulated expression of target genes. We have identified a window period when TCR A 75 A 75
stimulation induced FoxP3 expression in the bona fide T-regs. We have sorted CD4+
A5075
T‑cells in the PBMCs into CD25highFoxP3-, FoxP3highCD25high, CD25-FoxP3dim and CD2550
FoxP3- populations by flow cytometry. Genomic and proteomic study of T-regs and control 2550
populations of T-cells will enable the identification of the molecular processes that control
25
0
T-regs function and development.
CD25+
CD25-­‐
25
B
Contribution to patient and donor care or service development
Allogeneic HSCT is a curative treatment for a range of haematological neoplasms. About
50% of patients receive HSCT develop Graft versus Host Disease (GvHD) following HSCT,
despite GvHD prophylaxis treatment which has other complications including infections,
delayed stem cell reconstitution and compromised Graft versus Leukaemia effect (GvL).
T-regs bring the promise of the differential effects on the prevention of GvHD while
permitting GvL.
Our work on identification, isolation, expansion and mechanistic study of T-regs will
facilitate the translation of this exciting advance in immunology to a novel clinical
intervention.
isola@on; (B) & (C
CD25+ and CD25-­‐ fra
staining of CD25+
isola@on; (B) & (C) A staining of CD25+ an
C
0
0
B
B
(A) Frequency of (A) Frequency of CD3+CD4+CD25+FoxP3+ cells in the CD3+CD4+CD25+
(A) Frequency of
CD25+ and CD25-­‐ frac@ons aAer GMP
isola@on; (B) & (C) A representa@ve
CD25+ and CD25
CD3+CD4+CD25+Fox
staining of CD25+ and CD25-­‐ frac@ons
CD25+
CD25+
CD25-­‐
CD25-­‐
C
C
Tregs for GvHD, Wei Zhang
Tregs f
Tregs for G
T-regs for GvHD, Wei Zhang
Research and Development Triennial Report 2009-12
55
Theme
1
7
Stem cells and immunotherapies
Developing antigen-specific adoptive cellular
immunotherapy for CMV and EBV
Dr Frederick Chen
[email protected]
CONTRIBUTING TEAM MEMBERS: Heather Beard, Guido Frumento and Mohammad Raeiszadeh
NHSBT-Stem Cell Immunotherapy is the largest provider of stem cell processing
in the UK. The Birmingham facility processes 200 allografts/year. Haematopoietic
Stem Cell Transplantation (HSCT) can cure many patients with haematological
malignancies but carries considerable risk of mortality from disease relapse,
infections and graft-versus-host disease. As these complications are characterised
by T-cell deficiencies, adoptive T-cell therapy (ACT) offers potential solutions. In
Birmingham we are developing ACT for viral infections such as cytomegalovirus
(CMV) and Epstein-Barr virus (EBV).
A phase I clinical trial in adoptive transfer of HLA-tetramer-selected donor CMVspecific T-cells to HSCT recipients achieved resolution of refractory CMV infection
in 89% of patients, and demonstrated the efficacy of directly transferred fresh
unmanipulated donor CMV-specific T-cells. Building on this ’first-in-man’ trial in
collaboration with Prof Paul Moss, University of Birmingham, a phase II randomised
controlled trial, ACE~ASPECT was launched in 2010. This multi-centre trial recruits
unrelated transplant patients at high risk of CMV reactivation. To minimise donor
risk and enable rapid access, CMV-specific T-cells are selected from donor stem
cell harvests and cryopreserved for later off-the-shelf use. CMV-specific T-cells are
selected with commercial HLA-multimers (Streptamers®, STAGE-GmbH, Germany).
ACE~ASPECT is funded by Leukaemia Lymphoma Research and Cell Medica™,
the trial sponsor. NHSBT Birmingham manufactures the therapeutic cells locally for
Birmingham and Cell Medica nationally as Cytovir CMV™. The Birmingham centre
provides centralised immune monitoring for trial patients.
EBV is associated with several malignancies including Hodgkin’s disease,
nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease (PTLD)
after HSCT and solid organ transplantation. As PTLD expresses EBV-derived antigens,
we plan to target these with EBV-specific T-cells isolated from EBV-positive blood
donors who will be recruited to a panel of pre-tested lymphocyte donors.
RECENT PUBLICATIONS
The prevention and treatment
of cytomegalovirus infection
in haematopoietic stem cell
transplantation. Tuthill M, Chen
FE, Paston S, et al. Cancer Immunol
Immunother, 2009
The kinetics of CMV specific CD4 and
CD8 T-cell reconstitution post stem
cell transplantation, Raeiszadeh M,
Beard H, Frumento G, et al. Abstr.
NHSBT R&D Conference 2011
Evaluating novel EBV epitopes for
stimulating and selecting EBV-specific
T-cells for adoptive immunotherapy,
Frumento G1,2, Long H2, Briggs D1 et
al. Abstr. NHSBT R&D Conference
2011
Cord Blood T-cells retain early
differentiation phenotype suitable
for immunotherapy after TCR gene
transfer to confer EBV-specificity,
Frumento G, Zheng Y, Aubert G,
et al., in press American Journal of
Transplantation 2012
Modification of T-cells with T-cell receptors (TCR) or chimeric antigen receptors (CAR)
directed against given antigens increases their potency as demonstrated by the
impressive activity against chronic lymphocytic leukaemia and melanoma. However,
evidence suggests in vivo efficacy depends on the persistence and replicative capacity
of T-cells that decreases with age, rendering T-cells from older patients less potent.
We therefore explored the utility of cord blood (CB) T-cells for ACT. EBV-specific TCR
were transduced into CB T-cells and their properties studied. In comparison to adult
T-cells, CB T-cells proliferate better and have longer telomeres and telomerase activity
indicating greater replicative capacity and suitability for immunotherapy. The NHSBTCB Bank has over 17,000 CB units which could be utilised for T-cell immunotherapy.
Contribution to patient and donor care or service development
Virus-specific T-cells targeting CMV and EBV are new cell therapies for treating
patients with CMV reactivation and PTLD. There is the possibility of extending ACT to
treat different EBV-associated malignancies and life-threatening EBV infections in rare
immunodeficiency syndromes. Clinical-diagnostic assays developed for the trial can
form the basis of new immuno-diagnostic flow cytometry service to monitor anti-viral
therapy. A panel of white cell donors will be established to provide cells for virusspecific ACT.
56
Research and Development Triennial Report 2009-12
Adoptive cellular therapy:
A transplant patient receiving T-cells.
Inset: a syringe containing a
suspension of T-lymphocytes.
Theme
Stem cells and immunotherapies 1
7
Stem cells in leukaemia
Dr Allison Blair
[email protected]
CONTRIBUTING TEAM MEMBERS: Charlotte Cox and Paraskevi Diamanti
The prospects for patients with leukaemia have improved significantly and
many people can now be cured with intensive therapy, including stem cell
transplantation. However, relapse rates remain high for patients who have
disease that is resistant to treatment and in patients where the transplants are
ineffective, often due to insufficient numbers of stem cells in the transplanted
material. We are studying the characteristics of leukaemia cells to understand the
differences between stem cells that can initiate leukaemia and haemopoietic stem
cells that produce normal blood cells.
We have studied samples from children with leukaemia to determine which
leukaemia cells may be responsible for causing relapse and to develop methods of
targeting these resistant cells. We demonstrated that some leukaemia cells, known
as leukaemia stem cells, can initiate and maintain the disease. Leukaemia stem cells
may be responsible for causing relapse, so it will be important to develop therapies
that can eliminate these cells to prevent further disease progression. We are using
in vitro and in vivo model systems of leukaemia to test the effects of current and
novel therapeutic agents and to develop therapies that can target these leukaemia
stem cells.
The response of leukaemia patients to the first few weeks of therapy is a good
indicator of whether their treatment needs to be intensified or can be reduced. This is
known as minimal residual disease (MRD) monitoring. We are investigating leukaemia
stem cells in an attempt to identify markers on these cells that can be used in MRD
tracking techniques. In this way it may be possible to improve disease monitoring and
detect relapse at an early stage.
RECENT PUBLICATIONS
Cox CV, Diamanti P, Evely RS,
Kearns PR, Blair A. Expression of
CD133 on leukemia initiating cells in
childhood ALL. Blood 2009; 113(14):
3287-3296
Diamanti P. and Blair A. Stem cells
in childhood acute lymphoblastic
leukemia: identifying the most
relevant targets for therapy. Blood
2009; 113(18): 4477-4478
Diamanti P, Cox CV, Blair A.
Comparison of childhood leukemia
initiating cell populations in NOD/
SCID and NSG mice. Leukemia
2012;26(2):376-380
Group webpage
http://www.bristol.ac.uk/
cellmolmed/stem-cells/
haemopoietic
In order to provide improved therapeutic products
for these patients we are attempting to expand the
number of haemopoietic stem cells that are present
in donated cord blood. Cord blood is a readily
available source of cells for transplantation but the
number of stem cells is low and this has restricted
its use to mainly paediatric patients. Increasing the
number of stem cells transplanted should result in
replacement of the diseased leukaemia cells with
normal blood cells and improve survival and quality
of life for patients.
These approaches should improve current monitoring
and treatment options for patients.
Contribution to patient and donor care or
service development
The in vitro culture system we developed to support
proliferation of acute lymphoblastic leukaemia cells in
vitro has been implemented by the Regional Genetics Laboratory in the
South West to improve the quality and quantity of cytogenetic analyses
of ALL cells for routine diagnostic screening. The system we developed
enables faster, more efficient cytogenetic analysis, which is more likely
to result in an accurate karyotype.
Testing effects of drugs on leukaemia cells
Left panels: confocal images of leukaemia cells five
hours following drug treatment, showing reduced
green fluorescence in the treated sample.
Right panels: two months after treatment, leukaemia
cells cannot be detected in treated samples.
Research and Development Triennial Report 2009-12
57
Theme
1
8
Molecular and tissue engineering
Development of 2nd and 3rd generation tissue grafts
Professor John Kearney
[email protected]
CONTRIBUTING TEAM MEMBERS: Paul Rooney, Akila Chandrasekar and Richard Lomas
Tissue Services has continued to develop second generation, decellularised
tissue grafts owing to their reduced immunogenicity and their potential for
recolonisation by recipient cells. For the most promising tissues, the basic research
has been followed by a programme of translational research.
Decellularised human dermis has been clinically evaluated as a treatment for chronic
non-healing leg ulcers. This treatment has been shown to improve the ulcers and
in many cases lead to complete healing. Further studies will evaluate the clinical
performance of the dermis in other indications including severe burns and rotator cuff
injury. Clinical trials of decellularised cardiovascular tissues are currently being planned.
Although decellularised tissue allows recolonisation of the tissue matrix by the
recipient’s cells, there are clinical situations where progenitor or stem cells are very few
in number, which would lead to significant delays in recolonisation. We have therefore
begun to develop third generation grafts where the recipient cells are encouraged
to recolonise and differentiate in vitro prior to implantation. We have shown that
decellularised dermis can be repopulated with recipient fibroblasts and keratinocytes
to produce an autologous-like skin graft with a well formed epidermis and stratum
corneum (see Figure 1). For this study we used progenitor cells however stem cells
have been evaluated for other tissue matrices.
Studies with human mesenchymal stem cells have demonstrated that cyclic
compressive strain alone induces differentiation into osteogenic or chondrogenic
lineages depending on the applied strain.
RECENT PUBLICATIONS
Shortt A et al. The effect of amniotic
membrane preparation method on its
ability to serve as a substrate for the
ex-vivo expansion of limbal epithelial
cells. Biomaterials 2009; 30(6):105665.
Michalopoulos E et al. Development
of methods for studying the
differentiation of human
mesenchymal stem cells under cyclic
compressive strain. Tissue Eng. Part C,
2012; 18(4):252-62.
Wilshaw SP et al. Development and
characterisation of acellular allogeneic
arterial matrices. Tissue Eng. Part A,
2012;18(5-6):471-83.
Kearney J N et al. Engineering of
human tissue grafts. In Tissue and Cell
Processing an Essential Guide,
Wiley-Blackwell, 2012; 310-326.
Further studies have evaluated whether limbal stem cells can be combined with
decellularised matrices to provide a mechanism to replace missing cornea and hence
restore sight.
Contribution to patient and donor care or service development
Decellularised dermis does not elicit an immune response and is capable of becoming
repopulated with recipient cells. During the clinical evaluation, the dermis was
repopulated with recipient fibroblastic cells and blood vessels and a new epidermis
formed over the tissue, leading to complete healing of the wound. This successful
safety/efficacy evaluation now allows us to launch decellularised dermis as a new
Tissue Services product in 2012 in accordance NHSBT Strategic Plan for targeted new
product development and issue within Tissue Services.
Figure 1. Re-population of decellularised dermis with dermal
fibroblasts and keratinocytes.
58
Research and Development Triennial Report 2009-12
Figure 2. Appearance of cultured neoskin.
Theme
Molecular and tissue engineering 1
8
Tissue Development Laboratory –
from research to service
Dr Paul Rooney
[email protected]
CONTRIBUTING TEAM MEMBERS: Mark Eagle, Penny Hogg, Alasdair Kay and John Kearney
Over the past three years, the Tissue Development Laboratory (TDL) has
concentrated on developing new tissue allograft products and on improving
existing allografts based on in-house work and data coming through from
our research collaborators. The TDL has been instrumental in adding two new
allograft products into the range of grafts available to surgeons in the UK;
we have achieved one patent and have improved protocols for skin and bone
processing. In addition, members of the lab have successfully brought in income
for NHSBT by performing contract manufacturing work for two biotechnology
companies.
RECENT PUBLICATIONS
Wilshaw SP, Rooney P, Berry H et al.
Development and characterisation of
acellular allogeneic arterial matrices.
Tissue Engineering 2012; 18:471-483
Rooney P and Kearney J. New
developments in Regenerative
Medicine, Blood and Transplant
Matters, 2011; 33: 22-23
We have successfully developed a sterilised, de-cellularised dermis, which shows up to
98% removal of donor DNA without affecting any of the biological, biomechanical or
biochemical properties of the tissue (Figure 1). This process has been translated into
GMP tissue processing and is currently being assessed in a clinical setting.
Hogg P, Rooney P and Kearney J.
Development of a decellularised
dermis. Cell and Tissue Banking 2012,
E-pub ahead of print
Similarly, we have developed a method of producing demineralised bone matrix
(DBM) from the shafts of long bones and again this has been translated into GMP
processing. The DBM has been made into pastes and putties and is capable of
inducing new bone formation in an animal model (Figure 2); a clinical assessment is
planned to begin mid-2012.
Baylis O, Rooney P, Figueiredo F
et al. Epithelial outgrowth from
human cadaveric limbal explants
on human amniotic membrane is
delayed by the duration of corneal
organ culture. J. Cellular Physiology,
2012 e-pub ahead of print
We have patented a method of washing and sterilising intact femoral head bone
and part of the process has been incorporated into an improved method of washing
deceased donor bone, which is now supplied to surgeons. In addition, we have
developed methodology for cooling whole femoral condyles from knee joints to
-50oC without freezing or damaging the cells and we are continuing to develop a
method of storing these living, articular cartilage allografts long-term for issuing to
surgeons ‘off the shelf’ for transplant procedures as required.
More rapid bone repair
We have expanded our collaborations to include Queen’s University of Belfast and
Cambridge University and are currently working on making bioactive bone cement,
which incorporates DBM and/or human collagen, and which does not generate heat
as it cures. This cement may stimulate enhanced and more rapid bone repair when
used in orthopaedic procedures.
The TDL has also worked very closely with the new Medicines and Health Products
Regulatory Authority (MHRA) licensed Liverpool Advanced Therapy Unit to provide
expertise and facilities for the validation and translational work required to bring
cellular therapy products to clinical trials.
Two new tissue allografts have been introduced into the repertoire of grafts available
to surgeons. De-cellularised dermis can be used to treat burns, non-healing ulcers,
rotator cuff injuries and in breast reconstruction. The de-cellularised matrix can be
repopulated with recipient cells and become incorporated into the recipient with the
potential to grow and repair as part of the recipient. Demineralised bone matrix will
be used to enhance repair and new bone formation, as it is made into a paste and
putty, it can be moulded and pressed into the size and shape of a defect.
Figure 2. A and B, DBM paste inside of and being extruded from a syringe; C, new bone
formation induced when DBM is implanted into a muscle. BM = bone marrow; DBM =
remnants of implanted DBM; Ob = newly differentiated osteoblasts forming new bone.
Figure 1. Appearance of cells and nuclei
in cellular skin and cellular dermis and
absence of nuclei in decellularised dermis
(DCD). H&E and DAPI stains.
A
B
C
Research and Development Triennial Report 2009-12
59
Theme
1
8
Molecular and tissue engineering
Creating new blood vessels for tissue repair
Professor Suzanne Watt
[email protected]
CONTRIBUTING TEAM MEMBERS: Anita Mistry, Grigorios Tsaknakis, Cheen Khoo, Jen Kean, Dan Markeson,
Enca Martin-Rendon and Laura Newton
Cardiovascular diseases and delayed wound healing are enormous burdens to
both patients and healthcare systems, with expenditure exceeding £16 billion in
the UK alone. Such diseases and injuries may benefit from regenerative medicine
approaches. The human skin (dermis) presents an ideal model for tissue repair,
because it is thin, contains a microcirculation rather than a macrocirculation,
and there are FDA approved dermal substitutes in clinical use. One focus of our
laboratory is therefore on skin repair following burn injuries, in resistant non-healing
chronic wounds or in inherited/congenital skin conditions.
Although allografts or autografts and biologics have been used, autologous full
thickness skin grafts are currently the best means of resurfacing wounds, but, where
these are unavailable, artificial bio-engineered skin substitutes should prove equally
effective. A range of bioengineered skin constructs exists, yet there remains no ideal
composite skin substitute for permanent wound closure.
However, tissue engineered grafts often fail in the clinic because they do not develop a
sufficiently rapid blood supply. Key challenges include achieving early revascularisation
of the graft, identifying the best cells and/or biologics for optimal re-vascularisation and
determining their potency in different individuals, enhancing vascular cell numbers and
efficacy in revascularisation for enhanced skin repair, while limiting wound contracture
and scarring, and selection of optimal skin construct.
In addressing these challenges, we have assessed the ability of vascular stem/progenitor
cells (endothelial and mesenchymal) from different pre- and post-natal sources to
form blood vessels both in vitro and in vivo and have identified molecules contributing
to improved vessel formation in 2D and 3D cultures using functional readouts
(collaboration with Dr Maria Roubelakis, Academy of Sciences in Athens).
We have also developed a biobank of vascular cells with colleagues in the University of
Oxford (Professors Keith Channon and Hugh Watkins, and Dr Paul Leeson) for molecular
and genetic studies from individual matched donor umbilical cords. A key success has
been the development in collaboration with Professor Sheila MacNeil’s group (University
of Sheffield) of a clinical grade carrier for efficient transfer of mesenchymal stem cells,
which support blood vessel formation to decellularised human dermis in vitro.
RECENT PUBLICATIONS
Walker NG, Mistry A, Smith LE,
Eves PC, Foster S, Tsaknakis G,
Watt SM, MacNeil S. A chemically
defined carrier for the delivery of
human mesenchymal stem/stromal
cells to skin wounds. Tissue Eng.
2012;18:143-155
Athanassopoulos A, Tsaknakis G,
Newey SE, Harris AL, Kean J,
Tyler MP, Watt SM. Microvessel
networks in pre-formed in artificial
clinical grade dermal substitutes in
vitro using cells from haematopoietic
tissues. Burns 2012;38:691-701
Watt SM, Athanassopoulos A,
Harris AL, Tsaknakis G. Human
endothelial stem/progenitor cells,
angiogenic factors and vascular
repair. J.R.Soc.Interface 2010;7
Suppl 6:S731-S751
Zhang Y, Fisher N, Newey SE,
Smythe J, Tatton L, Tsaknakis G,
Forde SP, Carpenter L,
Athanassopoulos T, Hale SJ,
Ferguson DJ, Tyler MP, Watt SM.
The impact of proliferative potential
of umbilical cord-derived endothelial
progenitor cells and hypoxia on
vascular tubule formation in vitro*.
Stem Cells Dev. 2009; 18(2):
359-375
Contribution to patient and donor care or service development
Stem Cells and Immunotherapies (SCI) has established Human Tissue Authority (HTA)
Medicines and Healthcare Products Regulatory Authority (MHRA)
imp licensed laboratories, a requirement for manufacturing advanced
Improving Cord Blood Transplant Outcomes by
therapeutic medicinal products (ATMPs) for human application.
Understanding the Bone Marrow Niche
Additionally, as part of an EU Cascade consortium, standardised
European Good Manufacturing Production (GMP) protocols have
already been developed for defining manufacturing and release
criteria for human Mesencymal Stem/Stoma Cells and for taking these
cells to clinic. These developments together with our research on
wound healing will provide the necessary infrastructure to take these
approaches into clinical practice for the benefit of NHS patients.
Mesenchymal stem/stromal cells (MSCs) grow on an acrylic acid coated
carrier and can be transferred to human decellularised dermis as a prelude
to treating chronic non healing wounds (carried out in collaboration with
Prof. Sheila MacNeill)
60
Research and Development Triennial Report 2009-12
Theme
Molecular and tissue engineering 1
8
The work of the Clinical Biotechnology Centre in
supporting molecular therapy trials
Dr Paul Lloyd-Evans
[email protected]
CONTRIBUTING TEAM MEMBERS: Joan McColl, Denise Phillips, Antony Wright, Amanda Keyes, Jillian Harris, Tracy
Hutcherson, Helena Pastor, Karen Burks & Keith Smith (QA) and Simon Ellinson (Business Development Unit)
The Clinical Biotechnology Centre (CBC) located at the University of Bristol,
Langford is an integral part of the NHSBT research strategy for service and
product development. Its dedicated facility is well equipped and designed,
and combines the expertise of both NHSBT quality management and Good
Manufacturing Practice (GMP) trained scientific staff to operate in a highly
regulated environment. Our MHRA license has enabled us to specialise in
the small scale manufacture and importation of novel gene therapy and
recombinant protein investigational medicinal products for clinical trials in the
EU. To date it has manufactured over 40 gene therapy products and five novel
proteins for trials and is identified as an invaluable national resource whose
work is of clear relevance to NHSBT business. It remains an integral part of the
recently National Institute for Health Research (NIHR) funded NHSBT Molecular
and Tissue Engineering programme led by Professor Amit Nathwani (University
College London (UCL) and NHSBT) and are collaborators on a £1.2 million NIHR
Efficacy and Mechanisms Evaluation grant with Professor Christian Ottensmeier
(Cancer Sciences, Faculty of Medicine, Southampton) for a clinical trial into
haematological malignancy.
Over the past three years the CBC has diversified its services and products by
developing new manufacturing processes, offering external Quality Control (QC)
testing services and by varying its Medicines and Health products Regulatory
Authority (MHRA) license to realise other business opportunities. A GMP process
for conjugating monoclonal antibodies for radio-immunotherapy in the treatment
of leukaemia was introduced as well as a platform process for the production
of recombinant proteins from bacteria. A significant strategic partnership with
the world renowned University College London Gene Therapy Consortium was
established in 2011 when UCL’s new GMP facility was adopted onto NHSBT’s MHRA
license. In addition the CBC is providing ongoing quality management support and
process development expertise, along with the provision of
a pipeline of DNA vectors in support of its viral vector gene
therapy programme. The CBC is playing its part in the ongoing
Absorbance
gene therapy trials for the treatment of Cystic Fibrosis with the
Units
UK Cystic Fibrosis Gene Therapy Consortium and for Professor
Amit Nathwani’s highly acclaimed viral vector trial for the
treatment of Factor IX haemophilia. The CBC is providing
regulatory support for the importation of clinical trial materials
from the USA for delivery to the clinical trial centres involved.
RECENT PUBLICATIONS
Amit C. Nathwani, et al. AdenovirusAssociated Virus Vector – Mediated
Gene Transfer in Hemophilia N Engl J
Med 2011; 365:2357-2365
Figure 1. Purification of a plasmid DNA
vaccine by liquid chromatography in the
clean room.
Plasmid DNA
Gel Analysis
Purified
pDNA
OC
SC
1500 bp
Throughout 2011-12 the CBC has been working closely with
Professor David Anstee’s Group in growing artificial red cells
in a GMP bioreactor as part of initial scale-up and feasibility
studies for potential clinical studies in due course.
Contribution to patient and donor care or service development
The CBC is part of an important network of close to patient GMP facilities within
the organisation and an ideal setting for the development of new biological
medicinal products, specifically focused at introducing life changing therapies for
patients in the major NHS priority areas. Manufactured products are currently in
clinical trials for the treatment of a broad range of diseases including haematological
malignancies, prostate cancer, Cystic Fibrosis and HIV with up and coming trials for
graft versus host disease and rheumatoid arthritis opening soon.
Figure 2. Chromatogram of purified
plasmid DNA (pDNA) vaccine from a
bacterial lysate.
Research and Development Triennial Report 2009-12
61
Awards
Theme 1and honours 2009-11
Professor David Anstee
Editorial Board of Blood
Dr Lesley Bruce
British Blood Transfusion Society Kenneth Goldsmith (2010)
Dr Nick Burton
British Blood Transfusion Society Race and Sanger Award
(2010)
Professor Dave Collett
Member of Editorial Board of Statistical Methods in Medical
Research
Dr Geoff Daniels
Secretary General of the International Society for Blood
Transfusion June 2010 – present
Sub-Editor of Vox Sanguinis
Editorial board of Transfusion
Editorial board of Blood Reviews
Chair of the ISBT Working Party on Red Cell
Immunogenetics and Blood Group Terminology until
June 2010
President of British Blood Transfusion Society until
September 2009
Dr Lars Dölken
Robert Koch Post-Doctoral Prize (2011)
Rachel Johnson
British Transplantation Society Council member
Member of Editorial Board of Transplantation
Dr Enca Martin-Rendon
Editor of the Cochrane Heart Group, 2012
Professor Mike Murphy
Associate Editor, Transfusion Medicine Reviews. 2012
Elected Board Member, American Association of Blood
Banks 2010-12
Claes Högman Lecturer of Transfusion Medicine. Swedish
Society of Transfusion Medicine. 2009
British Computer Society and Computing UK IT Awards.
Winner, Public Sector Project of the Year. ‘BloodTrack’. 2009
Health Service Journal Awards. Winner, Improving Care
with Technology category. ‘Transformational improvement
in bedside clinical practice using wireless enabled bedside
technology’. Shortlisted for Secretary of State’s Award for
Excellence in Healthcare Management. 2009
Patient Safety Awards. Shortlisted for Technology and
IT to Improve Patient Safety category. Electronic Blood
Transfusion System. 2009
Healthcare Quality Improvement Partnership (HQIP)
Clinical Audit Awards. Shortlisted for Patient Safety Award.
National Comparative Audit of Blood Transfusion. 2010
Editorial Board Membership:
Transfusion
Transfusion Medicine
Expert Reviews in Hematology
62
Research and Development Triennial Report 2009-12
Dr Cristina Navarrete
Elected Councillor of the International Histocompatibility
Workshop
Dr Paul Rooney
Member of the Editorial Board of Cryobiology
Member of the Editorial Board of Cell and Tissue Banking
Professor Marion Scott
Chair of the Federation for Healthcare Science from
January 2008 to March 2010
Board Member of Medical Education England from
February 2009 to March 2010
Board Member of the Association of Clinical Scientists from
April 2009 to present
Dr Ashley Toye
Member of the Steering Committee for the European Red
Cell Society
Dr Dominic Summers
British Transplantation Society Roy Calne Award (2011)
Dr Stephen Thomas
British Blood Transfusion Society Race and Sanger Award
(2011)
Professor Suzanne Watt
Editorial Board of Current Tissue Engineering, 2012
Editorial Board of Niche, 2012
Editorial Board of Stem Cells and Development, 2009
1
PublicationsTheme
2009-11
Publications 2009
ACRE trial Collaborators. Effect of “collaborative
requesting” on consent rate for organ donation:
randomised controlled trial (ACRE trial). BMJ 2009
Oct 8;339:b3911. doi: 10.1136/bmj.b3911.:b3911.
Alabraba E, Nightingale P, Gunson B, Hubscher S,
Olliff S, Mirza D, et al. A re‑evaluation of the risk
factors for the recurrence of primary sclerosing
cholangitis in liver allografts. Liver Transpl 2009
Mar;15(3):330‑40.
Brunskill SJ, Hyde CJ, Stanworth SJ, Doree CJ,
Roberts DJ, Murphy MF. Improving the evidence
base for transfusion medicine: the work of the UK
systematic review initiative. Transfus Med 2009
Apr;19(2):59‑65.
Brunskill SJ, Hyde CJ, Doree CJ, Watt SM,
Martin‑Rendon E. Route of delivery and baseline
left ventricular ejection fraction, key factors of
bone‑marrow‑derived cell therapy for ischaemic
heart disease. Eur J Heart Fail 2009 Sep;11(9):887‑96.
Albanyan AM, Murphy MF, Harrison P. Evaluation
of the Impact‑R for monitoring the platelet storage
lesion. Platelets 2009 Feb;20(1):1‑6.
Cardigan R, Philpot K, Cookson P, Luddington
R. Thrombin generation and clot formation in
methylene blue‑treated plasma and cryoprecipitate.
Transfusion 2009 Apr;49(4):696‑703.
Albanyan AM, Harrison P, Murphy MF. Markers of
platelet activation and apoptosis during storage ‑ and
buffy coat‑derived platelet concentrates for 7 days.
Transfusion 2009 Jan;49(1):108‑17.
Cardigan R, Williamson LM. Production and storage
of blood components. In: Practical Transfusion
Medicine, editors Murphy M & Pamphilon D, 3rd
Edition (2009), Blackwells, London.
Albanyan AM, Murphy MF, Rasmussen JT, Heegaard
CW, Harrison P. Measurement of phosphatidylserine
exposure during storage of platelet concentrates
using the novel probe lactadherin: a comparison
study with annexin V. Transfusion 2009
Jan;49(1):99‑107.
Casals‑Pascual C, Idro R, Picot S, Roberts DJ, Newton
CR. Can erythropoietin be used to prevent brain
damage in cerebral malaria? Trends Parasitol 2009
Jan;25(1):30‑6.
Allain JP, Belkhiri D, Vermeulen M, et al.
Characterization of occult Hepatitis B virus strains
in South African blood donors. Hepatology 2009;
90:1868-1876
Anderson CA, Massey DC, Barrett JC, Prescott NJ,
Tremelling M, Fisher SA, et al. Investigation of Crohn’s
disease risk loci in ulcerative colitis further defines
their molecular relationship. Gastroenterology 2009
Feb;136(2):523‑9.
Anstee DJ. Red cell genotyping and the future
of pretransfusion testing. Blood 2009 Jul
9;114(2):248‑56.
Avent ND, Martinez A, Flegel WA, Olsson ML,
Scott ML, Nogues N, et al. The Bloodgen Project
of the European Union, 2003‑2009. Transfus Med
Hemother 2009;36(3):162‑7.
Barrett JC, Lee JC, Lees CW, Prescott NJ, Anderson
CA, Phillips A, et al. Genome‑wide association study
of ulcerative colitis identifies three new susceptibility
loci, including the HNF4A region. Nat Genet 2009
Dec;41(12):1330‑4.
Beckman N, Nightingale MJ, Pamphilon D. Practical
guidelines for applying statistical process control to
blood component production. Transfus Med 2009
Dec;19(6):329‑39.
Boralessa H, Goldhill DR, Tucker K, Mortimer AJ,
Grant‑Casey J. National comparative audit of
blood use in elective primary unilateral total hip
replacement surgery in the UK. Ann R Coll Surg Engl
2009 Oct;91(7):599‑605.
Brown AC, Hallouane D, Mawby WJ, Karet FE,
Saleem MA, Howie AJ, et al. RhCG is the major
putative ammonia transporter expressed in the
human kidney, and RhBG is not expressed at
detectable levels. Am J Physiol Renal Physiol 2009
Jun;296(6):F1279‑F1290.
Bruce LJ. Hereditary stomatocytosis and cation‑leaky
red cells‑recent developments. Blood Cells Mol Dis
2009 May;42(3):216‑22.
Bruce LJ, Guizouarn H, Burton NM, Gabillat N,
Poole J, Flatt JF, et al. The monovalent cation leak
in overhydrated stomatocytic red blood cells results
from amino acid substitutions in the Rh‑associated
glycoprotein. Blood 2009 Feb 5;113(6):1350‑7.
Creary LE, Girdlestone J, Zamora J, Brown J,
Navarrete CV. Molecular typing of HLA genes using
whole genome amplified DNA. Transfusion 2009
Jan;49(1):57‑63.
Dalton HR, Bendall RP, Keane FE, Tedder RS,
Ijaz S. Persistent carriage of hepatitis E virus in
patients with HIV infection. N Engl J Med 2009 Sep
3;361(10):1025‑7.
Daniels G, Castilho L, Flegel WA, Fletcher A, Garratty
G, Levene C, et al. International Society of Blood
Transfusion Committee on terminology for red blood
cell surface antigens: Macao report. Vox Sang 2009
Feb;96(2):153‑6.
Daniels G. Lutheran. Immunohematology
2009;25(4):152‑9.
Daniels G, Finning K, Martin P, Massey E. Noninvasive
prenatal diagnosis of fetal blood group phenotypes:
current practice and future prospects. Prenat Diagn
2009 Feb;29(2):101‑7.
Daniels G, van der Schoot CE, Gassner C, Olsson
ML. Report of the third international workshop on
molecular blood group genotyping. Vox Sang 2009
May;96(4):337‑43.
Caskey FJ, Johnson RJ, Fuggle SV, Start S, Pugh D,
Dudley CR. Renal after cardiothoracic transplant:
the effect of repeat mismatches on outcome.
Transplantation 2009 Jun 15;87(11):1727‑32.
Daniels G. The molecular genetics of blood group
polymorphism. Hum Genet 2009 Dec;126(6):729‑42.
Chapman CE, Stainsby D, Jones H, Love E, Massey E,
Win N, et al. Ten years of hemovigilance reports of
transfusion‑related acute lung injury in the United
Kingdom and the impact of preferential use of male
donor plasma. Transfusion 2009 Mar;49(3):440‑52.
Davison KL, Dow B, Barbara JA, Hewitt PE, Eglin
R. The introduction of anti‑HTLV testing of blood
donations and the risk of transfusion‑transmitted
HTLV, UK: 2002‑2006. Transfus Med 2009
Feb;19(1):24‑34.
Chaffe, B, Jones J, Milkins C, et al. UK Transfusion
Laboratory Collaborative: Recommended minimum
standards for hospital transfusion laboratories.
Transfus Med. 2009; 19; 156-158
Day E, Best D, Sweeting R, Russell R, Webb K,
Georgiou G, et al. Predictors of psychological
morbidity in liver transplant assessment candidates:
is alcohol abuse or dependence a factor? Transpl Int
2009 Jun;22(6):606‑14.
Charlton MR, Wall WJ, Ojo AO, Gines P, Textor S,
Shihab FS, et al. Report of the first international
liver transplantation society expert panel
consensus conference on renal insufficiency in liver
transplantation. Liver Transpl 2009 Nov;15(11):S1‑34.
Chimma P, Roussilhon C, Sratongno P,
Ruangveerayuth R, Pattanapanyasat K, Perignon JL,
et al. A distinct peripheral blood monocyte phenotype
is associated with parasite inhibitory activity in acute
uncomplicated Plasmodium falciparum malaria. PLoS
Pathog 2009 Oct;5(10):e1000631.
Clewley JP, Kelly CM, Andrews N, Vogliqi K,
Mallinson G, Kaisar M, et al. Prevalence of disease
related prion protein in anonymous tonsil specimens
in Britain: cross sectional opportunistic survey.
BMJ 2009 May 21;338:b1442. doi: 10.1136/bmj.
b1442.:b1442.
Collett D, Sibanda N, Pioli S, Bradley JA, Rudge C. The
UK scheme for mandatory continuous monitoring
of early transplant outcome in all kidney transplant
centers. Transplantation 2009 Oct 27;88(8):970‑5.
Compston LI, Li C, Sarkodie F, Owusu-Ofori S, et
al. Prevalence of persistent and latent viruses in
untreated patients infected with HIV-1 from Ghana,
West Africa. J Med Virol 2009; 81: 1860-1868.
Cooper SC, Aldridge RC, Shah T, Webb K, Nightingale
P, Paris S, et al. Outcomes of liver transplantation
for paracetamol (acetaminophen)‑induced hepatic
failure. Liver Transpl 2009 Oct;15(10):1351‑7.
Coste J, Prowse C, Eglin R, Fang C. A report on
transmissible spongiform encephalopathies and
transfusion safety. Vox Sang 2009 May;96(4):284‑91.
Cox CV, Diamanti P, Evely RS, et al. Expression of
CD133 on leukemia initiating cells in childhood ALL.
Blood 2009; 113(14): 3287-3296.
Dendrou CA, Plagnol V, Fung E, Yang JH,
Downes K, Cooper JD, et al. Cell‑specific protein
phenotypes for the autoimmune locus IL2RA using a
genotype‑selectable human bioresource. Nat Genet
2009 Sep;41(9):1011‑5.
Desai M, Neuberger J. Chronic liver allograft
dysfunction. Transplant Proc 2009 Mar;41(2):773‑6.
Diamanti P. and Blair A. Stem cells in childhood acute
lymphoblastic leukemia: identifying the most relevant
targets for therapy. Blood 2009; 113(18): 4477-4478.
Dudley CR, Johnson RJ, Thomas HL, Ravanan R,
Ansell D. Factors that influence access to the national
renal transplant waiting list. Transplantation 2009 Jul
15;88(1):96‑102.
Dunn C, Peppa D, Khanna P, Nebbia G, Jones
M, Brendish N, et al. Temporal analysis of early
immune responses in patients with acute
hepatitis B virus infection. Gastroenterology 2009
Oct;137(4):1289‑300.
Ellory JC, Guizouarn H, Borgese F, Bruce LJ, Wilkins
RJ, Stewart GW. Review. Leaky Cl‑HCO3‑ exchangers:
cation fluxes via modified AE1. Philos Trans R Soc
Lond B Biol Sci 2009 Jan 27;364(1514):189‑94.
Erdmann J, Grosshennig A, Braund PS, Konig IR,
Hengstenberg C, Hall AS, et al. New susceptibility
locus for coronary artery disease on chromosome
3q22.3. Nat Genet 2009 Mar;41(3):280‑2.
Finning K, Martin P, Daniels G. The use of maternal
plasma for prenatal RhD blood group genotyping.
Methods Mol Biol 2009;496:143‑57.
Flatt JF, Bruce LJ. The hereditary stomatocytoses.
Haematologica 2009 Aug;94(8):1039‑41.
Research and Development Triennial Report 2009-12
63
Publications 2009-11
Flegel WA, von Z, I, Doescher A, Wagner FF,
Strathmann KP, Geisen C, et al. D variants at the
RhD vestibule in the weak D type 4 and Eurasian D
clusters. Transfusion 2009 Jun;49(6):1059‑69.
Forsythe J, Cardigan R. Advisory committee on the
safety of blood, tissues and organs. Transfus Med
2009 Apr;19(2):57‑8.
Francis JJ, Stockton C, Eccles MP, Johnston M,
Cuthbertson BH, Grimshaw JM, et al. Evidence‑based
selection of theories for designing behaviour change
interventions: using methods based on theoretical
construct domains to understand clinicians’ blood
transfusion behaviour. Br J Health Psychol 2009
Nov;14(Pt 4):625‑46.
Francis JJ, Tinmouth A, Stanworth SJ, Grimshaw
JM, Johnston M, Hyde C, et al. Using theories of
behaviour to understand transfusion prescribing in
three clinical contexts in two countries: development
work for an implementation trial. Implement Sci 2009
Oct 24;24(4):70.
Ganesh SK, Zakai NA, van Rooij FJ, Soranzo N, Smith
AV, Nalls MA, et al. Multiple loci influence erythrocyte
phenotypes in the CHARGE Consortium. Nat Genet
2009 Nov;41(11):1191‑8.
Ghevaert C, Rankin A, Huiskes E, Porcelijn L,
Javela K, Kekomaki R, et al. Alloantibodies against
low‑frequency human platelet antigens do not
account for a significant proportion of cases of
fetomaternal alloimmune thrombocytopenia:
evidence from 1,054 cases. Transfusion 2009 Oct;
49(10):2084‑9.
Gillies M, Chohan G, Llewelyn CA, Mackenzie J,
Ward HJ, Hewitt PE, et al. A retrospective case note
review of deceased recipients of vCJD‑implicated
blood transfusions. Vox Sang 2009 Oct;97(3):211‑8.
Girdlestone J, Limbani VA, Cutler AJ, Navarrete CV.
Efficient expansion of mesenchymal stromal cells
from umbilical cord under low serum conditions.
Cytotherapy 2009;11(6):738‑48.
Hearnshaw S, Brunskill S, Doree C, Hyde C, Travis S,
Murphy MF. Red cell transfusion for the management
of upper gastrointestinal haemorrhage. Cochrane
Database Syst Rev 2009 Apr 15;(2):CD006613.
Hermans C, Wittevrongel C, Thys C, Smethurst
PA, van GC, Freson K. A compound heterozygous
mutation in glycoprotein VI in a patient with
a bleeding disorder. J Thromb Haemost 2009
Aug;7(8):1356‑63.
Hess JR, Sparrow RL, van der Meer PF, Acker JP,
Cardigan RA, Devine DV. Red blood cell hemolysis
during blood bank storage: using national quality
management data to answer basic scientific
questions. Transfusion 2009 Dec;49(12):2599‑603.
Hill AJ, Zwart I, Tam HH, Chan J, Navarrete C,
Jen LS, et al. Human umbilical cord blood‑derived
mesenchymal stem cells do not differentiate into
neural cell types or integrate into the retina after
intravitreal grafting in neonatal rats. Stem Cells Dev
2009 Apr;18(3):399‑409.
Hill AJ, Zwart I, Samaranayake AN, Al‑Allaf F,
Girdlestone J, Mehmet H, et al. Rat neurosphere cells
protect axotomized rat retinal ganglion cells and
facilitate their regeneration. J Neurotrauma 2009
Jul;26(7):1147‑56.
Hill QA, Hill A, Allard S, Murphy MF. Towards better
blood transfusion‑recruitment and training. Transfus
Med 2009 Feb;19(1):2‑5.
64
Hollyman D, Stefanski J, Przybylowski M, Bartido
S, Borquez‑Ojeda O, Taylor C, et al. Manufacturing
validation of biologically functional T‑cells targeted to
CD19 antigen for autologous adoptive cell therapy. J
Immunother 2009 Feb;32(2):169‑80.
Hustinx H, Poole J, Bugert P, Gowland P, Still F,
Fontana S, et al. Molecular basis of the Rh antigen
RH48 (JAL). Vox Sang 2009 Apr;96(3):234‑9.
Ijaz S, Vyse AJ, Morgan D, Pebody RG, Tedder RS,
Brown D. Indigenous hepatitis E virus infection in
England: more common than it seems. J Clin Virol
2009 Apr;44(4):272‑6.
Johnson RJ, Clatworthy MR, Birch R, Hammad A,
Bradley JA. CMV mismatch does not affect patient
and graft survival in UK renal transplant recipients.
Transplantation 2009 Jul 15;88(1):77‑82.
Jones CI, Bray S, Garner SF, Stephens J, de BB,
Angenent WG, et al. A functional genomics approach
reveals novel quantitative trait loci associated with
platelet signalling pathways. Blood 2009 Aug
13;114(7):1405‑16.
Jones CI, Garner SF, Moraes LA, Kaiser WJ, Rankin
A, Ouwehand WH, et al. PECAM‑1 expression and
activity negatively regulate multiple platelet signalling
pathways. FEBS Lett 2009 Nov 19;583(22):3618‑24.
Jones MN, Armitage WJ, Ayliffe W, Larkin DF,
Kaye SB. Penetrating and deep anterior lamellar
keratoplasty for keratoconus: a comparison of graft
outcomes in the United Kingdom. Invest Ophthalmol
Vis Sci 2009 Dec;50(12):5625‑9.
Kathiresan S, Voight BF, Purcell S, Musunuru K,
Ardissino D, Mannucci PM, et al. Genome‑wide
association of early‑onset myocardial infarction with
single nucleotide polymorphisms and copy number
variants. Nat Genet 2009 Mar;41(3):334‑41.
Khoo CP, Valorani MG, Brittan M, Alison MR, Warnes
G, Johansson U, et al. Characterization of endothelial
progenitor cells in the NOD mouse as a source
for cell therapies. Diabetes Metab Res Rev 2009
Jan;25(1):89‑93.
King MJ, Bruce L, Whiteway A. The mutant
erythrocyte band 3 protein in Southeast Asian
ovalocytosis does not bind eosin‑5‑maleimide. Int J
Lab Hematol 2009 Feb;31(1):116‑7.
Kirwan M, Beswick R, Vulliamy T, Nathwani AC,
Walne AJ, Casimir C, et al. Exogenous TERC alone
can enhance proliferative potential, telomerase
activity and telomere length in lymphocytes from
dyskeratosis congenita patients. Br J Haematol 2009
Mar;144(5):771‑81.
Kitchen AD, Hewitt PE. HIV screening reactivity due
to donor participation in HIV vaccine trials. Vox Sang
2009 Aug;97(2):169‑71.
Lamikanra AA, Theron M, Kooij TW, Roberts DJ.
Hemozoin (malarial pigment) directly promotes
apoptosis of erythroid precursors. PLoS One 2009
Dec 24;4(12):e8446.
Lawrie AS, Albanyan A, Cardigan RA, Mackie IJ,
Harrison P. Microparticle sizing by dynamic light
scattering in fresh‑frozen plasma. Vox Sang 2009
Apr;96(3):206‑12.
Lee E, Redman M, Owen I. Blocking of fetal K
antigens on cord red blood cells by maternal anti‑K.
Transfus Med 2009 Jun;19(3):139‑40.
Lefrere JJ, Hewitt P. From mad cows to sensible blood
transfusion: the risk of prion transmission by labile
blood components in the United Kingdom and in
France. Transfusion 2009 Apr;49(4):797‑812.
Research and Development Triennial Report 2009-12
Lemnrau AG, Cardoso S, Creary LE, Brown C, Miretti
M, Girdlestone J, et al. Human platelet antigen
typing of neonatal alloimmune thrombocytopenia
patients using whole genome amplified DNA
and a 5’‑nuclease assay. Transfusion 2009
May;49(5):953‑8.
Lenci I, Alvior A, Manzia TM, Toti L, Neuberger
J, Steeds R. Saline contrast echocardiography in
patients with hepatopulmonary syndrome awaiting
liver transplantation. J Am Soc Echocardiogr 2009
Jan;22(1):89‑94.
Li KK, Neuberger J. Recurrent nonviral liver
disease following liver transplantation. Expert Rev
Gastroenterol Hepatol 2009 Jun;3(3):257‑68.
Li KK, Neuberger J. The management of patients
awaiting liver transplantation. Nat Rev Gastroenterol
Hepatol 2009 Nov;6(11):648‑59.
Llewelyn CA, Wells AW, Amin M, Casbard A,
Johnson AJ, Ballard S, et al. The EASTR study: a new
approach to determine the reasons for transfusion
in epidemiological studies. Transfus Med 2009
Apr;19(2):89‑98.
Lok CY, Merryweather‑Clarke AT, Viprakasit V,
Chinthammitr Y, Srichairatanakool S, Limwongse C,
et al. Iron overload in the Asian community. Blood
2009 Jul 2;114(1):20‑5.
Lowe DP, Cook MA, Bowman SJ, Briggs DC.
Association of killer cell immunoglobulin‑like
receptors with primary Sjogren’s syndrome.
Rheumatology (Oxford) 2009 Apr;48(4):359‑62.
Martin‑Rendon E, Snowden JA, Watt SM. Stem
cell‑related therapies for vascular diseases. Transfus
Med 2009 Aug;19(4):159‑71.
Massey E, Paulus U, Doree C, Stanworth S.
Granulocyte transfusions for preventing infections
in patients with neutropenia or neutrophil
dysfunction. Cochrane Database Syst Rev 2009 Jan
21;(1):CD005341.
Meisinger C, Prokisch H, Gieger C, Soranzo N,
Mehta D, Rosskopf D, et al. A genome‑wide
association study identifies three loci associated
with mean platelet volume. Am J Hum Genet 2009
Jan;84(1):66‑71.
Mells G, Mann C, Hubscher S, Neuberger J.
Late protocol liver biopsies in the liver allograft:
a neglected investigation? Liver Transpl 2009
Aug;15(8):931‑8.
Mells G, Neuberger J. Long‑term care of the
liver allograft recipient. Semin Liver Dis 2009
Feb;29(1):102‑20.
Metcalfe P, Allen D, Kekomaki R, Kaplan C, de
HM, Ouwehand WH. An International Reference
Reagent (minimum sensitivity) for the detection
of anti‑human platelet antigen 1a. Vox Sang 2009
Feb;96(2):146‑52.
Mijovic A, Pamphilon D, Watt SM. Stem cell
transplantation and cellular therapy. In: Contreras M,
editor. ABC of transfusion medicine. London: BMJ
Books, Wiley‑Blackwell; 2009. p.104‑9.
Morley SL. Red blood cell transfusions in acute
paediatrics. Arch Dis Child Educ Pract Ed 2009
Jun;94(3):65‑73.
Murphy MF, Navarrete C, Massey E. Donor screening
as a TRALI risk reduction strategy. Transfusion 2009
Sep;49(9):1779‑82.
Publications 2009-11
Murphy MF, Staves J, Davies A, Fraser E, Parker R,
Cripps B, et al. How do we approach a major change
program using the example of the development,
evaluation, and implementation of an electronic
transfusion management system. Transfusion 2009
May;49(5):829‑37.
Murphy MF, Allard S, Newland AC. Modernizing
hospital transfusion laboratory services. Transfus Med
2009 Aug;19(4):153‑5.
Nathwani AC, Cochrane M, McIntosh J, Ng CY, Zhou
J, Gray JT, et al. Enhancing transduction of the liver
by adeno‑associated viral vectors. Gene Ther 2009
Jan;16(1):60‑9.
Navarrete C, Contreras M. Cord blood banking:
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Patents 2009-12
Inventors
Enca Martin-Rendon and Professor Suzanne Watt
Subject
Method for evaluating angiogenic potential
Reference
GB1007159-5
Filing Date
29/04/2010
Inventors
Lee Carpenter and Professor Suzanne Watt
Subject
Cardiovascular cells
Reference
GB1121101-8
Filing Date
08/12/2011
Inventors
Professor John Kearney, Paul Rooney,
George Galea, Tom McQuillan
Subject
Bone washing process
Reference
GB2469205A
Filing Date
06/10/2010
Inventors
Professor David Anstee and Jan Frayne
Subject
Conversion of fetal hemoglobin
Reference
GB1200458-6
Filing Date
11/01/2012
Inventors
Enca Martin-Rendon and Professor Suzanne Watt
Subject
Angiogenic factors
Reference
GB1118024-7
Filing Date
19/10/2011
Research and Development Triennial Report 2009-12
73
74
Research and Development Triennial Report 2009-12
Research and Development Triennial Report 2009-12
75