What are Red Blood Cells?
Red blood cells (RBCs) are the most abundant component of blood (Dean, 2005). The biconcave and anucleated structure of mammillary RBCs enable them to play an essential role in transporting gases and nutrients throughout the body (NHLBI, n.d.). RBC membranes contain protein and sugar antigens, substances that can elicit an foreign immune response (Dean, 2005). The International Society of Blood Transfusion has identified 33 distinct blood group systems consisting of over 270 antigens. (Mitra et al., 2014; Rahfeld and Withers, 2020).
ABO Blood Group System
The ABO blood group system holds the greatest clinical significance in transfusions and transplantations as its antigens are the most immunogenic (Mitra et al., 2014). The four basic ABO blood types are A, B, AB, and O. The base structure of the ABO blood group system is the H-antigen of O-type blood; it is the precursor to the ABO antigens and present on all RBCs of the ABO system (Rahfeld and Withers, 2020). The A and B antigens on type A, B, and AB blood differ from the H-antigen in the addition of an extra sugar group using A-transferase (GTA) and B-transferase (GTB) for A and B antigens, respectively (Rahfeld and Withers, 2020)(Fig. 1).
Figure 1 - Basic Structure of A, B, and H antigens of the ABO Blood System (Rahfeld and Withers, 2020)
The body’s immune system develops antibodies against ABO blood group antigens foreign to the individual's RBCs (Dean, 2005). Blood incompatibility occurs when an individual receives blood containing antigens that it has antibodies for. This elicits a potentially life threatening immune response that can result in RBC agglutination and subsequent hemolysis (Rahfeld and Withers, 2020). This poses a critical bottleneck on blood supply as it restricts the available pool of donors. As the H antigen on type O RBC is not recognized by anti-A or anti-B antibodies, type O blood functions as an effective universal donor blood that can be transfused to any individual; thus are in great demand (Rahfeld and Withers, 2020).
Transfusion Medicine and the Blood Shortage Issue
Blood transfusions are an integral component of healthcare, used to replace blood that is lost or provide blood when one’s body cannot make it properly (NHLBI, n.d.). They are needed in both routine and emergency situations: after an injury, during surgery, and for people with blood related disorders such as anemia, sickle cell disease, hemophilia, or cancer (NHLBI, n.d.). Around 234 million major operations are performed worldwide each year, with 63 million people undergoing surgery for traumatic injuries, 31 million for treating cancers, and 10 million for pregnancy-related complications (WHO, 2010). Transfusions contribute to saving millions of lives each year, drastically improving patients’ life expectancy and quality of life (WHO, 2010).
The availability of blood for transfusions is regulated by the supply and demand of safe, transfusable blood. Fluctuations causing seasonal shortages and excesses are regularly observed given that the blood market operates without market prices and with limited shelf-life (Slonim 2014). Concerning long-term trends are also increasingly apparent: patient-donor specificity, decreasing donations rates among younger populations (Fig 2), contribute to low supply, while demand continues to rise (WHO, 2010). A blood shortage occurs when local blood supply fails to meet its demand, and is especially prevalent in aging populations where greater medical care is required, developing countries where chronic blood shortages are common due to limited health services and access to safe blood, and developed countries where complex blood intensive medical procedures are increasingly common(WHO, 2010).
The COVID-19 pandemic has in particular exacerbated the blood shortage issue worldwide due to decreased blood donations and blood bank staff workers (Stanworth et al., 2020). Using Taiwan and its recent surge in COVID-19 cases as an example, it has been reported that the country's blood supply has fallen to the lowest in 20 years (Taiwan News, 2021). Significant dropoff in donations have also been observed in the African region (Fig 3). The significance of blood transfusions in rescuing patients calls for more robust measures to ensure national blood supply levels can satisfy demands.
Figure 2 - Trends in Rate of First Time Donors in Taiwan from 2010-2019 (Taiwan Blood Services Foundation, 2019)
Figure 3 - Gap in Blood Donations before and after COVID in the African Region (Loua, 2021)
Great emphasis has been placed on the voluntary and altruistic nature of blood donations. However, the limitation this places on available blood supply has led some organizations to resort to incentives such as payment, gifts, or other benefits to attract donors and increase the donor pool. Paid or commercialized donors often donate blood regularly to a blood bank for an agreed fee or sell their blood to banks or patients’ families (WHO, 2010). However, blood from these donors may not be as reliable or safe. Studies have reported that paid donors have the highest prevalence of transfusion-transmissible infections (WHO, 2010). Voluntary non-paid donors often donate blood with the desire to help others and are not pressured by blood banks or their financial status, and thus are more likely to reveal medical conditions that would make them ineligible for donations (WHO, 2010). On the other hand, paid donors often live lifestyles that expose them to blood-borne infections and usually donate blood for financial gain, making them more likely to withhold information about their conditions to protect their income (WHO, 2010). Incentivizing and commercializing blood further raises ethical concerns as human blood donations should respect an individual’s rights to their own body and wellbeing, but a commercialized market may give way to exploitation (WHO, 2010). We discussed the ethics behind this topic in our bioethics roundtable, where more information can be found here. Reducing the tension between blood supply and commercialization can help improve the quality of donated blood.
Blood organizations often use advertisements as a communication strategy to recruit more blood donors. They are promoted and spread through social media, radio, and TV (Sumning et al., 2018; Martin-Santana et al., 2017). Organizations sometimes invite or partner with prominent influencers, celebrities, politicians, and athletes to reach a greater audience as they have the ability to influence the public (WHO, 2019). Such solutions, however, are temporary, and may not be able to keep up as the gap in supply and demand continues to grow. More fundamental approaches are needed to tackle blood shortages.
Artificial Blood Substitutes
Artificial blood made to act as a substitute for red blood cells are designed to transport oxygen and carbon dioxide throughout the body (Sarkar, 2008). If successful, they can be used for blood transfusions to alleviate the blood shortage issue. The ideal artificial blood product is safe and compatible with all blood types of the human body, is able to be sterilized to remove disease-causing viruses and microorganisms, and can be stored for over a year (Sarkar, 2008). There are currently two types of RBC substitutes being studied that differ in the way they carry oxygen: perfluorocarbon based and hemoglobin based.
Perfluorocarbon (PFC) based substitutes are completely synthetic and can be made without any biological materials (Moradi et al., 2016). They are more effective in dissolving oxygen than water or plasma, and can be easily sterilized as they are heat resistant (Moradi et al., 2016). However, they are insoluble in aqueous phase, thus are required to be solubilized using an emulsifying agent before being given to patients (Moradi et al., 2016).
Hemoglobin (Hb) based substitutes utilizes the natural method of carrying oxygen in RBCs. Instead of dissolving oxygen like PFC substitutes, oxygen covalently bonds to hemoglobin (Sarkar, 2008). Transmission of infections is a concern for Hb products as its sources are from biological materials (Moradi et al., 2016). Raw hemoglobin is also unstable in solution and breaks down into toxic compounds in the body (Sarkar, 2008). To overcome these problems, modifications to the hemoglobin molecule is needed; this can be done by chemically cross-linking molecules or using recombinant DNA technology (Sarkar, 2008). Additionally, current Hb substitutes only last in the human body for a day, while whole blood transfusions can last 34 days (Sarkar, 2008).
Artificial blood substitutes are still in preliminary stages of development and no products have been approved for use by the US FDA (Moradi et al., 2016). There have been several clinical trials done, but many proved unsuccessful due to negative patient side effects. Scientists still need to learn more about how natural RBCs function in order to produce a substitute with fewer side effects, increased oxygen-carrying ability, and longer life span in the human body (PHLBI, n.d.).
THE NEED FOR O TYPE BLOOD
O type blood is often in short supply and is in the highest demand, since it is the most common blood type and is used for emergency transfusions and for immune deficient infants (American Red Cross, n.d.). Type O negative blood can be transfused to patients with any blood type and is used when there is no time to determine a patient’s blood type. Type O positive blood is also especially needed as it is the most transfused blood type and can be given to all patients with Rh-positive blood type (American Red Cross, n.d.).
We developed a model to simulate the weekly supply and demand of blood using data from the Taiwan Blood Services Foundation. Over a simulated period of 800,000 years, we predicted that O type blood would be in shortage 9.98% of the time. Fig 4 highlights a specific simulated week where O type is in severe shortage. We found that even in a country like Taiwan, where the rates of blood donation are one of the highest in the world, there is still a considerable need for O-type blood.
Figure 4 - Levels of Blood Excess/Shortage by Blood Types in an example simulated week. Week of 9/17-9/23, with simulated numbers based on +/- 5% fluctuations from historical data (2014).
In times of decreasing blood supply, it is crucial to avoid situations like these where blood is readily available, but wasted due to incompatibility of transfusions. Our project aims to reduce this issue through eliminating incompatibility by converting all blood types to universal O type blood, increasing the amount of useful blood available for transfusions.
To alleviate the blood shortage issue, we aim to increase the supply of universal donor blood by enzymatically converting A, B, and AB blood types to O type, eliminating patient donor incompatibility. This approach preserves the inherent function of existing red blood cells, making it advantageous to synthetic substitutes.
We have identified 3 glycoside hydrolases that act as molecular scissors to cleave A and B RBC surface antigens: α-Galactosidase from Bacteriodes Fragilis that hydrolyses the terminal galactose residues of type B RBC antigens, α-N-Acetylgalactosaminidase from Elizabethkinga Meningiosepticum that hydrolyses the terminal N-acetylgalactosamine residue of type A RBC antigens, and Endo-β-Galactosidase from Streptococcus Pneumoniae that hydrolyses the antigen trisaccharide of both A and B type RBC antigens (Fig 5) (Rahfeld and Withers, 2020). Selection of these enzymes were made based on physicochemical properties including optimum temperature, pH, reactivity conditions, and efficiency.
Our recombinant enzymes will be housed in columns through bead-immobilization on a leukodepletion filter membrane as part of a modular, two-step blood conversion kit that ensures safety and sterility, and enables hands-off maneuvering. We envision our product being integrated in the processing step of the blood supply chain (Fig 6). Using our blood conversion technology, we hope to expand the pool of universal donors. More information about the implementation of our project into healthcare systems can be found here.
Figure 5 - Enzyme cleavage sites of α-N-Acetylgalactosaminidase, α-Galactosidase, and Endo-β-Galactosidase (Cabezas-Cruz, 2017)
Figure 6 - Overview of our blood type conversion kit
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