In the UK, the NHS transfuses around 2.1 million units of blood each year (NHS, 2015). This is most commonly for anaemia due to various reasons such as lack of iron, genetic diseases (for example sickle cell anaemia), thalassaemia and malaria (NHS, 2015). The current total cost of a blood transfusion from the source of the blood to the recipient receiving the transfusion, inclusive of all tests, is £170.14 for the first unit and £162.01 for each subsequent unit (National Institute for Health and Care Excellence, 2015). Blood transfusions cost the NHS £898 million each year. 66% of this is for the hospital stay of the recipient, but overall this still equates to 3-4% of the annual budget of the NHS (Varney & Guest, 2003). The main problems with our current system are the increase in demand due to the ageing population, and a lack of safety. This report will address the key problems in detail, identify and evaluate two potential stem cell sources for ex-vivo production, and address the future direction of the process.
a. Ageing population
The main problem with our current system is the increase in demand. The key factor contributing to this is the ageing population. Elderly people are often afflicted by anaemia, and do not recover as well from medical procedures, and there is a correlation between age and blood use – increasing almost exponentially with age. According to the World Health Organisation, in high income countries such as the UK, 76% of the total blood donations go to the over 65s (World Health Organisation, 2017). A study in 2016 asked all hospitals that were supplied with blood components to provide the clinical indication of all of the units transfused, as well as the age and sex of the recipients over the course of two weeks – one in February 2016 and one in May 2016 (Tinegate, et al., 2016). The median age of use was 69 years old which correlates with the theory that the ageing population will exert unprecedented demand on the NHS Blood and Transplant (NHSBT) service. Figure 1 shows the red blood cell (RBC) usage per 1000 of the population. It can be seen that over the age of 65, there is a large increase in the usage of RBCs. The other peaks can be attributed to neonatal health problems and usage around child-bearing age. Overall, the study measured 73.2% of the total donated units in week one and 73.4% of the total donated units in week two. The wastage was 1.5% therefore the data is only missing for around 25% of the total donated units. As can be seen in table 2, the highest proportion of the blood units transfused were used for medical indications rather than surgical. This suggests we should be focussing development on RBCs for the treatment of diseases such as anaemia and thalassaemia, rather than for surgical procedures. The results of the study reaffirm that the ageing population is going to become even more of a prominent problem, due to the clinical indications of blood use.
Indication Percentage of RBC units used (%)
Table 2 shows the percentage of blood donations by each age band of donors. It can be seen that the majority of blood is donated by the under 65s. This means that the over 65 age group is using the majority of our supplies, while only contributing 1.64% of donations. In addition to this, as can be seen in Table 3, the projected population of the UK suggests that there is going to be an increase in the proportion of people over the age of 65, and a decrease in those under 65. This means that while the need for blood is increasing, the pool of donors is decreasing. Current estimates are that demand will increase by at least 2-3% each year as a result of the ageing population (Greinacher, et al., 2017). We have developed some techniques to reduce the number of units used, such as giving patients with heart disease restrictive transfusions, allowing older blood to be transfused, and cell salvage during surgery. These methods have worked to an extent – 40, 000 units were saved between 2001 and 2006 (The University of Edinburgh, 2017) – but this is not enough to compensate for the increased demand. We need to come up with an innovative solution to this problem.
Age group (years) <18 18-24 25-44 45-64 >65
Percentage of total donations (%) 2.73 13.23 43.31 39.09 1.64
Year UK population 0-15 years (%) 16-64 years (%) 65 years and over (%)
2005 60, 413, 000 19.3 64.7 15.9
2015 65, 110, 000 18.8 63.3 17.8
2025 69, 444, 000 18.9 60.9 20.2
2035 73, 044, 000 18.1 58.3 23.6
2045 76, 055, 000 17.7 57.8 24.6
b. Blood type proportions
Another issue related to demand is the proportions of people with each blood type. With a diversifying population, it can be assumed that the current data in table 4 will soon change. Currently, the rarest blood types in the UK are those which are found in black, Asian and minority ethnic groups. While this means fewer people need these blood types, it also means that the number of people able to donate is smaller. As the population diversifies, it cannot be guaranteed that there will be enough donors from these groups to keep up with demand. The NHSBT has estimated that they will need 200, 000 new donors each year in order to keep up with current demand, let alone future demand, and it seems unlikely that there will be enough minority groups donating blood for there not to be a shortage of rare types. This is not an easy problem to fix. Receiving blood from the wrong ABO or Rhesus group can result in catastrophic haemolytic transfusion reactions, which could be fatal.
ABO blood group Percentage of donor population with this group (%)
a. What do we currently test for?
Currently in the UK we already test blood for markers of several infections. The main ones are Syphilis, Hepatitis (B, C and E), HIV and Human T-lymphotropic virus (NHS Blood and Transplant, 2017), but with every test there is a risk of false negative results, in which case recipients could be inadvertently infected with one of these diseases. The risks are low (less than 1 in 1 million for HIV, HBV and HBC, and less than 0.1% for bacterial infections) but the NHS guarantees a 100% right to safe blood and any risk at all of infection makes this guarantee impossible. In addition to this, there is a risk of transfusion transmitted infections (TTIs) from diseases we do not know about, or cannot test for. Two situations illustrate this point. The first is the contaminated blood scandal in the 1970s and 1980s in which thousands of people with haemophilia were infected with blood contaminated with HIV and hepatitis C. This was because, at least at first, officials were not aware of the presence of disease. The potential for this to happen today is not ruled out by advances in medicine. There is still a risk that diseases are being transmitted by blood donation and we just have not discovered them yet. The second situation is that in 2009, there were 68 TTIs identified, which we could not test for (Stramer, et al., 2009). Since then, more have been added to this list, showing that the problem is ever changing and day-to-day more diseases could be transmitted via blood donation.
b. Variant Creuzfeldt-Jakob Disease (vJCD)
Variant Creuzfeldt-Jakob Disease (vCJD) is a fatal neurodegenerative disease characterised by isoforms of the pathogenic prion, PrP (Sawyer, et al., 2015). The prions accumulate in the brain and when they reach high levels they cause irreversible damage to nerve cells, resulting in a variety of horrific symptoms. The symptoms include memory loss, changes in personality, loss of balance, blindness, jerking movements and progressive loss of brain function (NHS, 2015). In other countries such as the USA, anyone who spent time in the UK between 1980 and 1996 is ineligible for blood donation (American Red Cross, 2017). This is because the prevalence of bovine spongiform encephalopathy (BSE, or ‘mad cow disease’) was high at that time, and there is a risk that anyone who ate meat from a cow between those years could be infected with vCJD. As we have no functional assay for the disease, we are unable to tell if the donors are infected, particularly since the disease has an incubation time of up to 50 years (Collinge, et al., 2006). In this time, the donor could have infected several hundred people if he or she had been donating blood. The only way to diagnose if a person has vJCD is to rule out various other conditions such as Alzheimer’s disease, Parkinson’s and a brain tumour (NHS, 2015), but at the moment the only way to provide a concrete diagnosis is to carry out a biopsy of the brain during the post mortem. Currently, the NHSBT is trying to reduce the risk of vCJD transmission as it does not accept donations from people who have had a blood transfusion since 1980, but there is still a risk that the donor could be infected from ingesting infected meat. As mentioned previously, the NHS guarantees 100% safe blood but clearly this is not possible with regards to vCJD. While there have only been 177 cases of vCJD disease up to 2015 (Urwin, et al., 2016), the projected number of carriers is significantly higher than this, and the number of carriers could perhaps be as high as in 1 in 2000 people in the population (Gill, et al., 2013).
One assay is currently being developed for vCJD. It detects the presence of disease-specific, abnormal PrP conformers (Sawyer, et al., 2015). It has been tested in CD-1 mice, and, as can be seen in Figure 2, it is possible to detect pre-clinical infection in CD-1 mice as early as 20 days post-innoculation. Obviously, this assay would need to be developed for use in human cells, but it gives promise to the likelihood of vCJD diagnosis and detection. While this assay could be used to screen all blood samples, it could also be used to aid ex-vivo RBC production. By screening one stem cell sample over many years, it can be ensured that it is free of vCJD and therefore safer to develop RBCs from than unscreened samples. Once we have a disease-free sample, we can amplify all of the RBCs from this singular sample. This is hugely advantageous over our current situation, but it will incur a high cost and has yet to be adapted for use in humans.
c. The Ixodes tick
The Ixodes (or deer) tick, transmits more diseases to humans than any other arthropod (Gulia-Nuss, et al., 2016). It is known to carry Borrelia burgdoferi which causes Lyme disease, of which there are 2000-3000 new cases each year in the UK (NHS, 2017). There are no documented cases of transfusion transmission of Lyme disease as B. burgdoferi has been proven to be unable to survive normal blood storage conditions (Ginzburg, et al., 2013). However, the Ixodes tick does carry Borrelia miyamotoi which causes relapsing fever and meningoencepalitis – meningitis with a progressive decrease in consciousness, cardiac and pulmonary complications and cranial neuritis – in the immunocompromised. It was identified 20 years ago but has only recently been considered to be a human pathogen. B. miyamotoi is capable of evading the immune system, and currently we have very limited antibody-based testing against the glycerophosphodiester phosodiesterase gene however homologues of this are present in other similar spirochetes (Krause, et al., 2015). We also know that B. miyamotoi can survive normal blood storage conditions (Thorp & Tonnetti, 2016). Figure 3 demonstrates this. All of the SCID mice injected with the components developed the disease after 21 and 42 days of storage. This shows that the pathogen can survive normal conditions, and that immunocompromised individuals are most at risk. This is especially problematic as the majority of blood recipients will fall into this category. In addition to this, some wild-type mice also developed the disease showing the potential for it to develop in healthy individuals. We need to develop an assay for the disease and have large population studies into the frequency of transmission and its seroprevalence in Lyme disease endemic areas. However, this would be very costly. Ex-vivo RBC production could solve this issue by screening a singular source for the pathogen and using this for all RBC expansion.
a. What makes an ideal stem cell source?
The ideal stem cell source should come from a source that would ordinarily be discarded in order to cut commercial costs, it should be non-immunogenic to reduce the risk of post-transfusion complications, it should have unlimited availability and, post-production, it should be fully functional as a RBC. There are four main stem cell sources but this report will focus on umbilical cord blood and induced pluripotent stem cells.
b. Umbilical cord blood
One potential stem cell source used for ex-vivo RBC production is cord blood stem cells. Cord blood is blood that remains in the umbilical cord and placenta after birth. Worldwide, over 780, 000 cord blood units are stored in over 130 private banks but there can only be used by the families which bank them. Only 400, 000 units are stored in 100 public banks which is a lot but over two times the amount is in private banks (Butler & Menitove, 2011). In the UK specifically, 22, 000 cord blood units are banked. 16, 000 are available for transplant and the rest can be used as research (NHS Blood and Transplant, 2017). This could be increased if only more people were informed about the option of freezing cord blood. It would normally go to waste so unless there are religious or medical reasons for not freezing it, why would we not encourage it? The blood can be cryopreserved for over 20 years but in this time, there is only a 0.04-0.005% chance that the child whose cord is banked will develop a disease treatable with their own cord blood by 21 years of age (Ballen, et al., 2008). This means the cord blood banked privately would go to waste, which is especially infuriating for people with rare blood types who may ordinarily struggle to find a match in the public system. Promoting public cord blood banking as an option, and developing ex-vivo production of RBCs from it, would help reduce the strain on the transfusion system, and leave those with rarer blood types much less vulnerable.
A 2017 study managed large scale generation of ex-vivo RBCs from cord blood CD34+ cells, and found that one human cord blood CD34+ cell could produce up to 200 million RBCs. This means that the yield from one unit of cord blood could be 500 blood transfusion units (Zhang, et al., 2017). After culturing, which took 21 days, the cells were stored for four weeks and had their haemoglobin levels monitored over this time. Figure 4 shows the results of this. It can be seen that, up to week four, the levels were maintained, indicating that the cells would be fully functional RBCs. Further testing would need to be done to deduce whether the cells could be stored for longer periods.
The main advantage of cord blood as a source of stem cells is the easy procurement with no risk or discomfort to the donors, and it would go to waste if not banked. It is also immediately available for use, and easily shipped (Newcomb, et al., 2007). In addition to this, there is a lower incidence of viral contamination compared to bone marrow donors. In a 2005 study, it was discovered that bone marrow donors had a viral infection frequency of 90% compared to 38.2% in umbilical cord blood donors (Behzad-Behbahani, et al., 2005). As previously stated, the cord blood can be stored for up to 20 years. There is also a reduced risk of graft versus host disease, which is one of the leading causes of mortality post-bone marrow transplantation. There is a high immune tolerance of umbilical cord blood cells because they are unable to generate cytotoxic T-Lymphocytes, which respond to allogenic antigens. Umbilical cord blood cells are also unable synthesize the pro-inflammatory cytokines interferon-gamma and tumour necrosis factor-alpha (Newcomb, et al., 2007). A relevant point to make is also that the use of cord blood is not associated with the same ethical issues as embryonic stem cells, for example, however some bodies may argue that the baby cannot consent to its cord blood being used so that may arise as an ethical problem with this source.
The main issue with cord blood is the fact that much of it is stored in private banks, so cannot be used medically. The best way to overcome the first problem is to promote cord blood donation as an option before birth. In this way, parents can be informed about the procedure and the benefits of storage in public banks. Other problems include a risk of viral infection, however as previously mentioned, this is less of a problem than with other sources. There is also a risk of the cord blood having an intrinsic genetic disease. This would have to be screened for and could end up increasing the cost but if we find one disease-free source we can use this for all expansions of RBCs. Currently, medical practises do not have the end goal of salvaging as many stem cells as possible. It seems unlikely that this would change without first having the ability to produce RBCs from stem cells. In US, the current cost of an umbilical blood stem cell transplant is $80, 000, but this is only to match the cost of a bone marrow transplant (Ballen, 2017). In reality, the true cost is likely to be much less than this.
c. Induced pluripotent stem cells (IPSCs)
Another potential stem cell source is induced pluripotent stem cells (IPSCs). They are cells which are reprogrammed from your own somatic cells using different transcription factors, which are integrated using various viral vectors. They have the unique properties of self-renewal and differentiation but without the ethical issues of other cell types (Singh, et al., 2015). The main advantages of IPSCs are their unlimited proliferation potential (Yamanaka & Blau, 2010), there is a low risk of immunorejection as they are made from the patient’s own somatic cells, and we could potentially reprogram somatic cells directly into RBCs by over-expressing suitable genes and turning fibroblasts into megakaryocytes (Zeuner, et al., 2012).
However, there are several downsides to using IPSCs. The first is the potential association with cancers. There are two systems used to integrate the transcription factors into somatic cells. The first group are Integrating Viral Vector Systems. This is where the viral genome is incorporated into the host cell genome (Singh, et al., 2015). This method is very efficient but there is a risk of cancer formation if the virus inserts itself into the wrong part of the genome, thus causing detrimental downstream effects. The second group, Non-Integrating methods, are not associated with the same cancer risks but the efficiency of the systems are very low. In general, the biggest problem with IPSCs is the low yield as this throws into question the whole feasibility of it as a source. IPSCs also have an incredibly low enucleation rate, increasing potential costs as the post-expansion modification would be more extensive (Dorn, et al., 2015).
Another point that needs to be considered is that the majority of IPSCs produce RBCs with foetal haemoglobin. In theory, this is less efficient than adult haemoglobin and we need to evaluate if it is worth spending millions of pounds developing this as a therapy for it to be less efficient than our current methods. However, some people with sickle cell disease, for example, do live with high levels of foetal haemoglobin in their blood. Research into this must be done before pressing forward with IPSCs as a potential source. An ethical problem that will be encountered is that some public bodies will not agree with reprogramming cells.
V. The SH2B3 gene
Genetic manipulation has been considered as an approach to resolving some of the cost and yield issues found with ex-vivo production. A 2016 study subjected 4678 individuals to whole exome sequencing, looking for allelic variants associated with enhanced RBC production. 19 of the individuals had a missense, or loss of function, variant in the SH2B3 gene on chromosome 12 associated with significantly increased haemoglobin levels and RBC counts compared to controls (Giani, et al., 2016). SH2B3 is a negative regulator of signalling downstream of cell surface receptors involved in erythropoiesis. This means it slows red blood cell production. Suppressing this gene, as in the allelic variant, leads to an increase in expression of erythropoiesis responsive genes, increasing red blood cell production. Knocking the gene out in stem cell lines had the potential to have the same effects ex-vivo, thus forming the basis for the most recent research.
Giani, et al. used lentiviral vectors to express two types of short hairpin RNAs (shRNAs), sh83 and sh84, which target SH2B3 in adult haematopoietic stem cells. Lentiviruses were used as they can integrate into both dividing and non-dividing cells. They are a vector which can integrate an RNA sequence (in this case an shRNA) into their genome which is reverse-transcribed within the cell to produce DNA. The DNA then inserts into the genome. When the gene is transcribed, the RNA produced associates with the RNA-induced silencing complex. This can detect mRNA with a complementary sequence and destroy it, thus silencing the gene (Moore, et al., 2010). After integrating the RNA into the genome, the cells were stimulated to undergo erythropoiesis and were compared to a control (shLuc, an empty vector). The results showed a 1.6-2-fold increase in enucleation, earlier maturation, increased haemoglobin and increased expansion in cell number. Figure 5 shows the expansion relative to the number of starting cells. It shows that cells with the shRNA undergo much more expansion than the control cells and that sh84 is possibly better than sh83 at increasing expansion. This may be due to sh84 knocking out more mRNA but overall the shRNAs are very similar in structure apart from a couple of bases so more research would be needed into this.
The next stage of the process was to find a method of large-scale application. Viral-mediated delivery is inappropriate for large-scale use as there is a risk of viral infection – a problem ex-vivo production is designed to overcome. An alternative solution is CRISPR-Cas9 mediated genome editing which is faster, cheaper and more accurate than other genome editing systems (US National Library of Medicine, 2018). This involves a small piece of RNA with a “guide” sequence binding to a specific target sequence (Ledford, 2016). It also binds to the Cas9 enzyme which causes a double stranded break in the DNA at the target location (in this case, exon 3 in SH2B3). This DNA is then repaired using the cell’s own repair system. Figure 6 shows that, compared to the wild type, the knockout cells had a higher proliferation rate meaning this is a potential method of increasing large scale expansion rates.
a. Recipients and Blood Types
My recommendation is that we should focus on providing ex-vivo blood for those people with rarer blood types. In this instance, it may be wiser to focus on developing O negative blood – which can be received by people with all blood types – and then, if successful, branch out into other subgroups. The blood should first be developed for use in patients with long-term conditions such as sickle cell anaemia and thalassaemia, and those who are immunocompromised. This is because these patients not only use the majority of our stored supplies, but are most at risk of death from infection due to transfusion, and therefore are more likely to benefit from the solutions ex-vivo production aims to provide.
In order to be researched by the NHS, therapies must be approved by the Health Research Authority. It is likely that some forms of stem cells, such as embryonic stem cells, are going to have more arguments against them than adult stem cells, for example. The ethical issues surrounding each stem cell type must be evaluated, along with the proliferation potential, in order to find the best source. I think the best source is likely to be cord blood stem cells. As they would go to waste anyway, they provide the advantages of easy recovery of cells and have no risk or discomfort to the donors. The main ethical issue with this is that the infant cannot consent to the use of the blood cells but, as they would not have a need for them, this may be less of an issue compared to other sources.
c. Future Amplification Methods
As stated previously, knocking out the SH2B3 gene has the potential to increase proliferation. We know this works in mouse stem cells so future research needs to focus on evaluating whether this works in human stem cells. This method is predicted to be 20% cheaper than other ex-vivo approaches, as well as using fewer starting cells. Future work could also include identifying other potential genes which we can combine with SH2B3 in order to fully enhance the proliferation potential of the stem cell. It could be beneficial to do another study, similar to the one which identified the SH2B3 gene. One study has used lentiviral transgenesis to insert a gene for erythropoietin into stem cells so they can produce their own cytokines, further reducing the cost of the process (Du & Zhang, 2010).
Access to safe blood is vital for our NHS to function properly. The demand has been increasing for many years and with the ageing population this is only going to become more of an issue. Adding the risk of infection into the situation, it is clear we need to come up with an alternative solution in order to have enough blood, and blood which is fit for purpose. Cord blood cells are likely to be the best source – they have the fewest ethical issues and the most potential to be a functional source. However, we need to ensure our amplification methods are cost effective and efficient. Before we do this, we need to have testing over many years to ensure our source is disease free. This must be done as the idea behind ex-vivo production is that it is safer than our current system. Ex-vivo production is not a quick, or cheap, solution to our current issues but with NHS investment and significant research I believe it could revolutionise our healthcare system.
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