7Biom018W Immunohaematology And Haemostasis Answers Assessment Answers
Haemopoietic stem cells can be found within the bone marrow, Umbilical cord or peripheral blood. For the culturing to occur there is a requirement of stem cells (SCs). Stem cells are undifferentiated and have the ability to self-renew. Pluripotent SCs can derive 200 different types of differentiated cells.
- Brief Function and importance of red blood cells.
- Emphasis on the need “in vitro” techniques in producing red blood cells.
- Brief explanation on its clinical prospects.
- Layout the things to be discussed in the main-body of the essay.
- Different ways to pursue to produce red blood cells in vitro, (Equipments etc…)
- Feasibility in regards to its production
- Its necessity for clinical use
- Addressing whether Red cell production improves clinical outcomes for patients.
- Address any contradicting studies towards invitro red cell production.
- Limitations in in vitro production of red blood cells.
- Future prospects of in vitro production of red blood cells.
- Necessity of producing in vitro red blood cells.
- Weigh studies in support or in contradiction to its clinical use.
- Emphasis to its relevance.
- Future implication.
Answer:
The in Vitro Process
Erythropoiesis is the process through which stem cells differentiate and proliferate in to mature red blood cells. In adults, this process of red blood differentiation produces more than 2 x 1011 erythrocytes in a day. The factors that promote proliferation and that which induce differentiation are always in opposition and therefore a balance is essential for the erythroid development, by using the stem cell factor and the erythropoietin (Xi et al, 2013). The erythropoietin, also called EPO, stimulates the synthesis of haemoglobin and also used in terminal synthesis. The EPO initiates the action of antiapoptotic proteins thus prevents the action of apoptosis on erythroid progenitor cells. Over the years researcher and scientists have shown a relationship between red blood cells development and factors such as glucocorticoids and insulin. Nakamura (2008) claims that there has been difficulties in assessing some signaling pathways in erythrocytes development in vivo as maturation and renewal processes in the bone marrow happen almost parallel. Despite too much research on the development of the red blood cells, some mechanisms and processes (such as enucleation and erythropoiesis) on molecular and cellular maturation and differentiation of red blood cells are unknown. Introduction of the in vitro model of red blood cells production helped exhibit every stage and details in their (RBCs) development (Porwit, McCullough & Erbe, 2011). The vitro model was a strong mechanism used to analyze and investigate the molecular tools and factors employed in differentiation, development and proliferation of RBCs.
Health cord is collected from volunteers, after obtaining informed consent, in to sterile tubes. Through the process of centrifugation the single nuclear cells are obtained. Purity has to be maintained above 95 percent. In a medium free from serum, one by ten power six per milliliter are cultivated for initial expansion. According to Miharada & Nakamura (2012), hematopoietic stem cells found in umbilical cord blood and bone marrow are the materials used in the production of the RBCs in vitro. They claim that the umbilical cord blood cells are sufficiently available as they are thrown away after parturition. The umbilical cord blood offers a dependable and useful source of red blood cells, without ethical and critical complications provided the mother has given her consent. In vitro, differentiation of these cells depends on cloning of EPO, creation of erythroidrestricted colonies and stem cell factor (Hoffman, 2012). The conditions responsible for maturation of red blood cells cannot be used to maintain proliferation of the cells according to an observation made by Fibach, about 20 years ago. Therefore he came up with a liquid culture system. This was a two-step process. In the first, cytokines and glucocorticoids are provided so as to enhance the proliferation process. In the second step, Fibach used EPO to help the red blood cells progenitors survive in their final stages. He also used the EPO to help in maturation of the RBCs precursors.
This two-step culture system has undergone advancements since it was introduced and has helped the synthesis of more mature red blood cells. The first step has undergone successive improvements such as replacing the conditioned media with SCF, EPO and lowly concentrated GM-CSF and IL3 and other cytokines (Douay, 2001). These cytokines were used to ensure that the red blood cells progenitors survived their late stages and also increase BFU-E number (Neildez-Nguyen et al. 2002). Stromal cells have also been used in vitro to expand multipotent and immature red blood cell progenitors (Fujimi et al. 2008). It was also discovered later that glucocorticoids and estradiol could be used to prevent maturation of the erythroid (Migliaccio et al. 2002). To improve the terminal maturation of the red blood cell precursors, thyroid hormone and insulin have been added to the EPO in the second step (Leberbauer et al. 2005). Another improvement that have taken effect in the past decade is addition of molecules to prevent further differentiation of the erythroid in this second step of in vitro (Miharada et al. 2006, Migliaccio et al. 2010). The figure below shows the process involved in in vitro synthesis of red blood cells
Necessity of the RBC for clinical use
According to Vas (2016), blood transfusion is among the most important procedures in health care settings. The need for blood transfusion arises in most cases due to medical emergencies and other conditions such as genetic haemolytic disorders. Since different individuals have different blood groups, a shortage of blood cells for transfusion is created. Other factors such as viruses, antigens and prions create risks of reactions in case of blood transfusion (Zimring et al. 2011). Migliaccio, Whitsett, Papayannopoulou and Sadelain (2012) claim that more than 40 percent of African-American sickle cell anemia patients experience immune reaction after being transfused with blood from Caucasian descent. Also, considering the fact that some blood groups are rare some geographical areas, there arose a call from researchers to find a way to make these blood groups evenly available (Murphy & Pamphilon, 2006). Researchers tried a couple of options such as chemically and enzymatically modified red blood cells to come up with some blood groups to no much success (Natanson et al. 2008). The in vitro haematopoiesis was used to solve this problem. Research on the in vitro haematopoiesis process showed that genetic substances and blood group antigens could be altered. Therefore the cultured red blood cells could be used in patients with blood disorders (like the sickle cell disease) and also for patients having rare blood groups.
Issues
According to Li, Wu, Fu & Han (2013), the in vitro synthesis of red blood cells has not yet taken deep roots in the health arena regardless of its benefits due to some ethical and clinical issues around their clinical utility. Ethical considerations, storage problems, errors in genetics and shelf life issues are big challenge in the in vitro process and more research is being done to tackle these.
Ethical considerations arise in the use of umbilical cord blood. The health professionals must obtain consent of the parents so as to use the umbilical cord blood as this brings in issues of generic data protection guidelines and should be handled with caution. According to Lee et al. (2016), about 1 x 106 CD34+ cells are collected from the umbilical cord. Baek et al. (2009) think that these cells collected are not enough amplify to attain transfusion needs. Cultured blood cells, according to Xie et al. (2014), have lower shelf lives than the cells present in donated blood. In their research, Giarratana et al. (2011) discovered that cRBCs and normal red blood cells almost have same half time, raising the question of if in vitro synthesis of red blood cells is worth the time and cost. Migliaccio et al. (2012) think that the in vitro process is challenging and very costly. It is proven that erythroid cells cannot proliferate unless at a concentration of more than 106, posing one of the major technical challenges. The in vitro process involves producing large volumes of red blood cells, with bioreactors being the only equipment used for this purpose in the biotechnology firm (Timmins & Nielsen, 2011). A good understanding of the final processes in the differentiation of erythroid enhances the in vitro process by decreasing the volume of media required. A clear understanding of the cell mediated factors, hormones, death pathways and cell adhesion inhibitors are also essential in this process (Kim & Baek, 2011). The final step of in vitro erythroid maturation needs stromal cells. The use of these cells affect the GMP production procedures since they are of animal origin (Kim & Baek, 2011).
Improvements and research on in vitro process
Earlier research on in vitro process discovered that human stem cells obtained using CD34+ biomarker could be used for safe blood transfusion without the risk of immune reactions, due to the ability of the cells to differentiate and replicate in to enucleated cells. However, it was discovered that these cells had a low shelf life and the process was at risk of transmitting infectious pathogens present in the blood group antigens thus making the blood unsafe for transfusion. This called for more research on the in vitro synthesis of RBCs. Among the results of this research was the discovery that use of F68 reduced the risk of haemolysis as well as preventing the infections which could be transmitted during the transfusion. This research also found a way of improving the maturity and shelf life of the erythrocytes. However, the RBCs transfusion had not yet been tested in human beings until the second decade of the 21st century. After a successful transfusion in to a health individual, the shelf life of the cRBCs was found to be 26 days. Since then, subsequence research have been conducted on ways to improve the in vitro red blood cells synthesis. In future the in vitro synthesis could be of great help to the patients suffering from sickle cell anaemia. The table below was discovered from the research and where the percentage concentration of cRBCs were recorded at various intervals. CPM is the RBC count per minute in the transfused individual and k is the value obtained at each interval. K is obtained by dividing CPM at each interval by CPM on day 0.
|
Time of collection |
CPM |
K=0.6 (29)% |
K= 1.2(29)% |
K=2.2 (29)% |
|
0 |
994±19 |
100 |
100 |
100 |
|
5 |
902±6 |
94 |
96 |
? 100 |
|
14 |
624±4 |
68 |
74 |
86 |
|
26 |
351±3 |
41 |
48 |
63 |
Conclusion
Red blood cells transfusion is a common therapy in hospitals, delivering millions of patients every year. This blood is obtained from donors which opens a risk to transmitting other infection s to the patient. The discovery of the in vitro process was a big step in the health sector. Bone marrow and umbilical cord cells are preferred due to their ability differentiate and replicate rapidly. After attaining consent of the parent, the umbilical cord blood is collected for the process. The in vitro synthesis of RBCs has reduced risks of antigen reaction and transmission of xenogeneic infections. Researcher have explored a range of materials like yolk cells, iPS and mononuclear cells. Despite the cord blood providing a better solution in in vitro process, there are issues that call for attention. These include ethical issue in handling of genetic materials and contamination risks. Currently, research embarks on coming up with a better option for co-culture material to promote the half-life and maturity of the RBCs.
References
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Leberbauer, C., Boulmé, F., Unfried, G., Huber, J., Beug H. & Müllner E. W. 2005. Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. The NCBI, Vol. 105, No. 1, pp. 85-94. Doi: 10.1182/blood-2004-03-1002
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