Artificial Cells, Blood Substitutes, and Biotechnology, 34: 589599, 2006 Copyright Q Informa Healthcare ISSN: 1073-1199 print/1532-4184

online DOI: 10.1080/10731190600974038

Genomics and Blood Substitutes for 21st Century Europe (EuroBloodSubstitutes)

Kenneth C. Lowe
School of Biology, University of Nottingham, University Park, Nottingham, UK

Abstract: Blood transfusion is a medical intervention practised throughout the world. Blood is a biologically active material that can transmit diseases (e.g., HIV=AIDS and, perhaps, vCJD). People are becoming increasingly concerned about blood safety, despite improved screening and processing. Consequently, they are reluctant to donate blood or receive transfusions. Such problems can be solved by the development and incorporation into transfusion practices of so-called blood substitutes to replace some blood uses. The EuroBloodSubstitutes Project is funded by the European Union Framework 6 Programme to develop a technological platform for producing novel haem proteins and blood substitute components using micro-organisms (bacteria, fungi, yeast) as cell factories. The Project will focus on bacteria (Escherichia coli), yeast (Pichia pastoris) and, longer-term, filamentous fungi (Aspergillus niger), all organisms used to synthesize commercially important products. The multi-centre Consortium consists of the Universities of (1) Nottingham (UK), (2) Essex (UK), (3) Denmark Technical (Denmark), (4) Lund (Sweden), (5) Milan (Italy), (6) Nancy (France), (7) Parma (Italy), (8) Rome La Sapienza (Italy), (9) Semmelweis (Hungary), together with (10) Alligator Bioscience, AB (Sweden), (11) LCC Engineering & Trading GmbH (Switzerland), (12) Scottish National Blood Transfusion Service (UK), and (13) Sanquin Bloodbank (The Netherlands). The EuroBloodSubstitutes Project will be informed by lay and professional stakeholders (e.g., clinicians,

EuroBloodSubstitutes is an acronym for a Specific Targeted Research Project (STREP) entitled Genomics and Blood Substitutes for 21st Century Europe (Contract No. 503023) supported by the European Union, 6th Framework Programme Priority (FP6-2002-LIFESCIHEALTH). It is a pleasure to acknowledge the expert administrative assistance provided by the Project Manager, Mrs. Bethany York. Address correspondence to Kenneth C. Lowe, School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. E-mail: kclowe@ nottingham.ac.uk 589

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blood donors, patient groups, prescribers and policy makers). Outcomes of the Project are (1) the production of an information pack, decision aids and physician training aids, giving balanced overviews of the benefits and risks of transfusion of blood or potential substitutes, and (2) an interactive web site (www.eurobloodsubstitutes.com) for information dissemination. This will improve knowledge and address misunderstandings about transfusion issues in a climate of changing patient expectations on blood safety and benefits of blood substitutes.

INTRODUCTION Blood transfusion is practised throughout the world. Blood is a biologically active fluid that can transmit diseases, especially HIV=AIDS and, perhaps, human variant Cruetzfeld-Jacob Disease (vCJD). People are becoming increasingly concerned about blood safety, despite improved screening and processing, and this is making them reluctant to donate blood or receive blood transfusions. Such problems can, in principle, be solved by developing injectable substitutes for blood. Whilst such products are commonly referred to as blood substitutes, they should perhaps be more accurately described as oxygen therapeutic drugs or oxygen therapeutics. Several authors have emphasized the benefits and shortcomings of current blood substitute products, including materials based on (1) the natural oxygen-carrying blood component, haemoglobin (Hb), processed from human or animal blood, (2) synthetic, gas-carrying fluorinated compounds [perfluorochemical (PFC) liquids], or (3) encapsulated Hbs [111]. There is much debate about the ideal characteristics of blood substitutes based on Hb. The older paradigm was that such products should have properties (oxygen affinity, viscosity, oncotic characteristics) that mimicked those of blood. The newer paradigm, postulated primarily by Winslow and colleagues [9], is that, to avoid unwanted vasoconstrictory side effects in recipients, Hb-based products should exhibit a higher oxygen affinity (lower P50 than blood), elevated oncotic properties (to maintain vascular volume) and a Hb content as low as possible (to limit toxicity and tissue arteriolar oxygen delivery). The basis of this new approach is to minimize extravasation of the Hb molecules and prevent over-oxygenation of tissue arterioles that would otherwise cause capillary vasoconstriction through the so-called autoregulation theory [9]. While it may seem, on first encounter, counterinitiative to suggest that new generations of Hb-based blood substitutes should be designed to be less effective at promoting tissue oxygenation, there is good evidence from experimental studies to indicate that this approach is feasible [9].

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THE EUROBLOODSUBSTITUTES PROJECT In 2004, a multi-centre, pan-European team, led by the University of Nottingham, initiated a new, 3-year research project, entitled Genomics and Blood Substitutes for 21st Century Europe (EuroBloodSubstitutes). The EuroBloodSubstitutes Project is funded by the European Union Framework 6 Programme to develop a technological platform for producing novel haem proteins and blood substitute components using micro-organisms (bacteria, yeast and filamentous fungi) as cell factories. The EuroBloodSubstitutes Project complements an on-going programme of research at the University of Nottingham, conducted in collaboration with other European and international institutions, that is focused on the development, properties, applications and incorporation into routine transfusion practices of blood substitutes to replace some blood uses. BLOOD SUBSTITUTES FROM CELL FACTORIES The EuroBloodSubstitutes Consortium will produce novel blood substitute components (haem proteins) using cell cultures. The project will focus on bacteria (Escherichia coli), yeast (Pichia pastoris) and, longerterm, filamentous fungi (Aspergillus niger). Such cultures are widely used as cell factories for synthesizing commercially-important products. The advantages of the chosen micro-organisms to the EuroBloodSubstitutes Project are: 1. E. coli was the first protein expression system to be used and is extremely well understood. It is excellent for the rapid and relatively inexpensive, large-scale production of many proteins giving high yield. 2. P. pastoris is less well studied but is becoming increasingly used for the expression of human recombinant proteins. It allows both intra- and extra-cellular protein production, typically at high yields. 3. A. niger is very well studied for the production of secreted proteins at high yields. A baseline for the production of human Hb from genetically modified micro-organisms [12] and plants [13,14] has been established. Such use of cells as factories for producing high-value human therapeutic proteins is, for plants at least, a rapidly expanding area of biotechnology [15]. This approach avoids using human or animal blood as starting material, thereby overcoming cultural and ethical objections to animal or human donor blood-derived Hb and the theoretical problem of transmitting

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infections. Such recombinant molecules are, in principle, virus free and contain no residual red blood cell membrane contaminants. In terms of the generation of novel haem proteins, one advanced product was OptroTM (Baxter, Deerfield, USA), containing cross-linked Hb (rHb1.1) and produced in E. coli through the expression of a modified human Hb gene. Clinical efficacy (Phase II) trials with OptroTM in surgical patients were completed in 1999. Other Hb mutants with altered oxygen and nitric oxide binding properties have also been produced [16]. Outstanding issues concerned with scale-up and purification costs of core recombinant proteins, together with identifying and overcoming bottlenecks in their production, will be addressed by the research activities of the EuroBloodSubstitutes Consortium. The EuroBloodSubstitutes Consortium consists of a team of experts in 13 academic and industrial institutions, which are: . . . . . . . . . . . . . University of Nottingham, Nottingham, UK University of Essex, Colchester, UK Technical University of Denmark, Lyngby, Denmark Henri-Poincare, Nancy, France Universite Semmelweis University, Budapest, Hungary Universita degli Studi di Milano, Milan, Italy Universita degli Studi di Parma, Parma, Italy University of Rome La Sapienza, Rome, Italy University of Lund, Lund, Sweden LCC Engineering & Trading GmbH, Egerkingen, Switzerland Alligator Bioscience AB, Lund, Sweden Scottish National Blood Transfusion Service, Edinburgh, UK Sanquin Bloodbank, Leiden, The Netherlands. A list of consortium partners and contact details is given in Appendix 1.

PARTICIPATION OF STAKEHOLDERS A novel aspect of the EuroBloodSubstitutes Project is that the research will be informed by lay and professional stakeholders (e.g., clinicians, blood donors, patient groups, prescribers, biotechnologists, industrialists and policy makers). The Project has a distinct workpackage component entitled Blood and Blood Substitutes in European Society that aims to gather quantitative and qualitative data on perceived benefit and risks associated with blood donation and transfusion. Such results will inform the strategic development of blood substitutes.

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Whilst the technical (biological) and, to a lesser extent, the economic aspects of risk in transfusion have been generally well studied, surprisingly little attention has focused on the perceptions of risk associated with the transfusion of blood, blood products or, most especially, potential blood substitutes. It is pertinent to ask (1) what relevant stakeholder groups know about transfusion, (2) how safe people perceive blood and blood products to be, (3) how the latter information might influence their own or others perceptions of risk linked to transfusion, and (4) the extent to which approved blood substitutes might be preferred over a persons own (autologous) or donated (allogeneic) blood. Information on what stakeholders perceive to be the benefits and risks of the receipt of blood and blood substitutes will inform future transfusion strategies.

Benefit and Risk Perceptions in Transfusion Medicine One of the earliest reports on public perceptions of the risk of blood transfusion was that of Paul Slovic and colleagues in 2000 [17]. Their study consisted of a telephone survey of 1,204 people in the USA in 199798. Overall, 36% of respondents did not believe the blood supply in the USA was safe and 33% of respondents indicated that they would not accept a transfusion of blood from a blood bank. There were significant gender differences, with women more strongly opposed to transfusion than men, and variations in opinions between members of different ethnic sub-groupings. A parallel programme of research in the UK studied the knowledge base and perceptions of risk of transfusion among key stakeholder groups, with the assessments focused on adult blood donors and non-donors, anaesthetists, general practitioners and healthcare journalists [1821]. The overall perception of the risk of transfusion across all respondents was low, compared to other hazards in society (e.g. smoking, exposure to X-rays), with infection being cited as the greatest concern. Moxey et al. [22] used a semi-structured survey instrument to examine the perceived risks of a transfusion with donor (allogeneic) blood, the preferences and willingness to pay for pre-donation of their own (autologous) blood and the desired role in the decision-making process in post-surgical patients, members of interest groups concerned with blood transfusion and the public in Australia in 20012002. Of the 206 respondents, 33% expressed concerns about receiving allogeneic blood transfusion but the risks of disease transmission were again perceived as being low. Importantly, autologous donation was perceived as removing all the risks associated with transfusion. However, autologous transfusion is costly and not applicable to all patients, especially

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those who are anaemic [23]. Furthermore, the increased protection against transmission of disease afforded by donating autologous blood is limited and may not justify the increased cost of this approach [24]. Studies in the UK [18,19] showed that, perhaps not surprisingly, physicians viewed transfusion as being less risky than laypeople, a finding subsequently confirmed by similar studies in Canada [25]. Interestingly, Rock and others [26] used a multiple choice questionnaire to evaluate differences in both the basic and clinical knowledge of physicians with different specializations in Canada. The mean score for clinical knowledge (63%) was significantly greater than that for basic knowledge (37%) and, perhaps predictably, haematologists and anaesthetists scored highest in both categories. There is, clearly, a case to be made for developing strategies for further improving physician knowledge on current transfusion issues, perhaps through the use of training aids, given that such professionals play a key front-line role in communicating with the public on healthcare issues.

MESSAGE FRAMING AND CUEING Experimental studies examining message framing [27] and cueing [28] on issues associated with blood donation and transfusion have also been conducted with young adults in Britain. In the former, participants were given printed materials containing identical factual information on blood safety presented either as a gain frame (i.e., lives saved), a loss frame (i.e., lives lost), or a combined frame (that is, a loss frame presented in a positive context). Overall, participants receiving gain frame information were more confident in blood safety. This emphasizes that the framing of the message on the safety of blood=blood substitutes influences the perceptions of stakeholders. In a further study, Farrell et al. [28] tested the hypothesis that providing contextual cues specific to HIV would cause survey respondents to express an increased belief that the virus can be contracted by donating blood. Adult subjects were randomly assigned into one of 3 experimental groups that were all given printed information about the small risk of infection from blood transfusion. The variation across the experimental conditions was the specific example of infection given, namely (1) data on the low risk of contracting hepatitis C through blood transfusion, (2) data on the low risk of contracting HIV through transfusion, or (3) no specific example of infection risk. When participants were subsequently asked the single question Do you think you could catch HIV by giving (donating) blood in the UK?, the HIV-cued group were almost 11 times more likely to answer yes than

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the hepatitis C-cue or no cue groups. This study shows that providing contextual cues on HIV increased the expressed belief that the virus could be contracted through donating blood. The authors concluded that future survey materials on blood safety should minimize contextual effects. This study further emphasized that the way in which information on blood safety is presented to people can have significant effects on survey responses. Design of Decision Aids and Communication Tools One useful way of communicating information on blood safety to prospective patients is through the use of decision aids to assist in making informed choices between transfusion options that might be available. In one such study, Grant and others [29] used decision aid, consisting of a booklet and audiotape, to help patients decide whether to pre-donate their own blood before open-heart surgery. If their answer was positive, they would then be transfused with autologous rather than donor blood during surgery, if needed. Initial questionnaires, completed prior to the use of the decision aid, evaluated patients knowledge, transfusion preferences, decisional conflict (i.e., amount of uncertainty about the course of action to be taken) and risk perceptions. Follow-up questionnaires were completed after the use of the decision aid. Overall, the decision aid improved knowledge and risk perceptions of blood donation and transfusion and, importantly, increased the percentage of patients willing to accept autologous transfusion. A further advantage of using decision aids is that they offer a multi-format approach for communicating information on healthcare issues that appeal to people with different educational backgrounds. Lee and Mehta [30] subsequently evaluated the impact of a visual risk communication tool on knowledge and perception of risk associated with blood transfusion among the Canadian public. Risk communication with both written and visual presentational formats increased knowledge of transfusion risk and decreased the perceived dread and severity of the transfusion risk. However, neither format changed the perceived knowledge and control of transfusion risk, nor the perceived benefit of transfusion. The authors recognized that risk communication materials using a multi-format approach may provide added value to patients. Analysis of Sources of Trustworthy Information A related study by Ferguson et al. [21] explored the types and sources of information that physicians and lay people considered as most reliable

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for information on transfusion risk. Perceived trustworthiness of different sources of information was related to what people think that they know about blood donation and transfusion (i.e., confidence in their knowledge) rather than what they actually know in terms of, for example, performance on a multiple choice quiz. This is important to ensure that factual information on the benefit and risk of blood and potential substitutes are targeted to a format most likely to be used as a source by stakeholders.

STRATEGY AND OUTCOMES OF THE EUROBLOODSUBSTITUTES PROJECT Many of the above approaches on engaging stakeholders on issues linked to blood donation and transfusion will be incorporated into EuroBloodSubstitutes Project. Field studies will target relevant groups of people, initially in the UK and then more broadly throughout Europe, to identify current issues in blood use in the context of biotechnological developments pertinent to the envisaged longer-term incorporation of blood substitutes into transfusion practices. Interestingly, a recent article in Forbes Global Magazine [31] discussed on-going clinical trials with the Hb-based blood substitute, PolyHeme (Northfield Laboratories Inc., Evanston, IL, USA). The article focused on attempts by Northfield of obtain regulatory approval for the drug as an alternative to blood (i.e., oxygen therapeutic) in trauma patients. As part of the next phase of the trials, the FDA required the company to conduct community consultations, including discussions with community groups about PolyHemes benefits and risks. This emphasizes the need for closer dialogue between stakeholders (manufacturers, prescribers and patients) as part of the formal acceptance and approval processes for blood substitutes. In addition to new knowledge and understanding of the feasibility of producing novel haem proteins and critical blood components from microbial cell factories, the outcomes of the EuroBloodSubstitutes Project will be (1) the production of a novel information pack, decision aids and physician training aids that give balanced overviews of the benefits and risks of transfusion with blood or a potential substitute, and (2) an interactive web site (www.eurobloodsubstitutes.com) for information dissemination. Overall, the EuroBloodSubstitutes Project aims to improve knowledge and address misunderstandings about current issues associated with blood donation and transfusion in a climate of changing patient expectations on blood safety and inadequate factual information on the benefits and risks of blood substitutes.

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APPENDIX 1 The Euro Blood Substitutes Consortium

University of Nottingham, School of Biology, Nottingham (UK) Dr. K. C. Lowe (Scientific Co-ordinator) Mrs. B. York (Project Manager) University of Essex, Department of Biological Sciences, Colchester (UK) Professor C. Cooper Technical University of Denmark, Process Biotechnology, Lyngby (Denmark) Professor J. Neisen Universite Henri-Poincare, Department of Physiology, Nancy (France) Dr. P. Menu Semmelweis University, Institute of Human Physiology and Experimental Research, Budapest (Hungary) Professor A. Eke Universita degli Studi di Milano, Dipartimento di Scienze Tecnologie Biomediche, Milan (Italy) Professor M. Perrella Universita degli Studi di Parma, Department of Biochemistry and Molecular Biology, Parma (Italy) Professor A. Mozzarelli University of Rome La Sapienza, Department of Biochemical Sciences, Rome (Italy) Professor A Bellelli University of Lund, Chemistry and Chemical Engineering, Lund (Sweden) Professor L. Bu low LCC Engineering & Trading GmbH, Egerkingen (Switzerland) Mr. W. Gletting Alligator Bioscience AB, Lund (Sweden) Dr. M. Andersson Scottish National Blood Transfusion Service, National Science Laboratory, Edinburgh (UK) Dr. C. Prowse Sanquin Bloodbank, Research and Development, Leiden (The Netherlands) Professor A. Brand

kclowe@nottingham.ac.uk bethany.york@nottingham.ac.uk

Ccooper@essex.ac.uk

in@biocentrum.dtu.dk

menu@pharma.uhp-nancy.fr

eke@elet2.sote.hu

michele.perrella@unimi.it

andrea.mozzarelli@unipr.it

andrea.bellelli@uniroma.it

Leif.Bulow@tbiokem.lth.se

willi.gletting@chemsupply.ch maa@alligatorbioscience.com

chris.prowse@snbts.csa.scot.nhs.uk

anneke.brand@bloodrtd.nl