1. Bio preservation of Red Cell Components 2. CULTURED RBCs 3. solvent plasma

1. Bio preservation of Red Cell Components
2. CULTURED RBCs
3. Solvent plasma

Hypothermic storage
Cryopreservation
Lyophilization

Desiccated RBCs
Three major sources of cells are under consideration:
circulating stem and progenitor cells from adults or from cord blood
immortalized progenitors
pluripotent stem cells.
Immortalized Progenitors

LIQUID CULTURE METHODS- by the SED (stem cell factor (SCF), erythropoietin, and dexamethasone) and STIF cocktails (stem cell factor, thombopoietin, insulin-like growth factor-2, fibroblast growth factor-2)
ENUCLEATION- separation of extruded nuclei from cRBCs
SCALING UP- using cord blood CD34+ cells in bioreactors
and treated for 4 h with TNBP (tri-nitrobutylphosphate)solvent and with Triton X-100 detergent

1. 1. Bio preservation of Red Cell Components 2. CULTURED RBCs 3. solvent plasma Presented By- Dr. Shiny Moderator- Dr. Nidhi Bansal

2. INTRODUCTION Biopreservation - P rocess of maintaining the integrity and functionality of cells held outside the native environment for extended storage times. The biopreservation of RBCs for clinical use can be categorized based on the techniques used to achieve biologic stability and ensure a viable state after long-term storage . 

3.  Current RBC B iopreservation approaches H ypothermic storage Cryopreservation L yophilization

4. HYPOTHERMIC STORAGE H ypothermic storage (1–6°C) of RBCs became feasible nearly a century ago, when Rous and Turner introduced the first glucose–citrate storage solution. Detrimental changes in RBC metabolism and biological function characterized with hypothermic storage. are commonly termed ‘the hypothermic storage lesion’ (HSL)

5. RBC HSLs include - the depletion of vital metabolites, such as ATP, 2,3 diphosphoglycerate and potassium cell senescence continuous decrease in pH loss of membrane components the release of microvesicles alterations in RBC rheological characteristics including filterability & deformability

6. Possible outcomes include- impaired tissue oxygenation inflammation transfusion-related immunomodulation intravenous hemolysis

7. CRYOPRESERVATION Red cells can be frozen and are stable for prolonged periods in a frozen state. Freezing of RBCs is mainly done to preserve units of blood with rare phenotypes or to build blood inventory for emergency use in disasters. Done with cryoprotective agent- Glycerol Glycerol- After entering the red cell, it provides an osmotic force that will stop water migration from the red cell due to extracellular ice formation.

8. Damage if frozen without cryoprotective agents- • Red cell dehydration: When extracellular water freezes before intracellular water, red cells collapse due to an osmotic pressure difference created between red cells and their surroundings, resulting in the diffusion of intracellular water out of the red cells. • Intracellular ice: When intracellular water freezes before extracellular water, red cells rupture owing to an increase in intracellular salt concentration drawing in more water and expanding the cell.

9. Frozen RBCs can be stored for 10 years. Rare frozen units may be used beyond the expiration date as literature reports satisfactory cell recovery and viability from units stored for up to 21 years. Deglycerolization - the unit is thawed at 37°C with gentle agitation taking about 10 minutes for complete thawing. Glycerol is removed gradually from thawed RBCs by washing with sterile saline solutions of decreasing osmolarity to avoid red cell haemolysis

10. Desiccated RBCs- Lyophilization Lyophilization (freeze-drying) involves the removal of most unbound water from biologic materials through controlled freezing followed by the sublimation of ice under vacuum . Effective lyophilization prevents sample shrinkage, minimizes chemical changes, and maintains product solubility to allow easy rehydration. 

11. Dry storage could potentially extend the product’s shelf life, reduce the weight and volume of the storing vehicle, facilitate transportation and save energy by eliminating the need for refrigeration. But u nfortunately, the majority of the attempts to stabilize RBCs in the dried state have resulted in poor cellular recovery , partially because of Hb oxidation . At present, no reconstituted RBCs meet the standards for transfusion.

12. CONCLUSION Currently, the only way to successfully preserve Hb for transfusion medicine applications is in its natural environment, the RBCs. Our ability, or inability, to preserve RBCs under various conditions (hypothermic, frozen or dry) is largely dictated by the oxidative state of Hb. Our success in preserving RBCs through hypothermic or frozen storage relies, at least in part, on keeping the Hb in its reduced state. Our failure to stabilize RBCs in the dried state or to create Hb-based oxygen therapeutics has been affected by our inability to avoid Hb-induced oxidative damage.

13. Other Newer Types of Components

14. CULTURED RED BLOOD COMPONENTS In-vitro production of red cells  Approximately 1% of the RBCs are eliminated from the circulation every day, maintenance of the average adult RBC mass of 2.5 × 10 13  requires the daily production of more than 200 billion RBCs. HSCs sequentially differentiate into common myeloid progenitors and megakaryocyte-erythroid progenitors and then into unipotent progenitors restricted to the erythroid lineage. 

15. Sources of cultured red cells Production of cRBCs for clinical purpose will require the identification of cell sources that can reliably be tapped and used to develop a highly scalable production procedure that recapitulates the erythropoiesis process Three major sources of cells are under consideration: circulating stem and progenitor cells from adults or from cord blood immortalized progenitors pluripotent stem cells.

16. Circulating Stem and Progenitor Cells With the best available methods, stem and progenitor cells found in 1 unit of cord blood can theoretically be expanded into more than 500 units of RBCs  This level of amplification is sufficient to significantly expand the blood supply of rare blood groups, which would be an important milestone for the field, and justifies investment in this technology. Drawback- their limited proliferation potential  Since these cells are not immortal, production of cRBCs remains dependent on the donation-based collection and testing system

17. Immortalized Progenitors  In general, cellular immortalization requires simultaneous stimulation of proliferation and inhibition of terminal differentiation and cell death Studies of murine erythroid precursors have led to the identification of several factors that control these processes in erythroid cells. These factors include GATA-1 , which promotes erythroid development , and PU.1 , which binds to GATA-1 and inhibits erythroid terminal differentiation I mmortalization of erythroblasts can be achieved by manipulating the expression of transcription factors, tumor suppressors, and nuclear receptors T his approach might be safe because cRBCs are not oncogenic since they are enucleated and remain fully functional after irradiation.

18. Pluripotent Stem Cells The third potential permanent source of cells for the production of cRBCs is pluripotent stem cells, such as human embryonic stem cells ( hESCs ) and induced pluripotent stem cells ( iPSCs ). The main advantages of these cells are that they are immortal and karyotypically stable and that they can be reproducibly generated from any individual with a variety of well-developed methods    Genetic engineering of pluripotent stem cells, which is technically possible, further expands the potential use of these cells as a source of cRBCs .

19. METHODS LIQUID CULTURE METHODS- by the SED ( stem cell factor (SCF), erythropoietin, and dexamethasone) and STIF cocktails ( stem cell factor, thombopoietin , insulin-like growth factor-2, fibroblast growth factor-2) ENUCLEATION- separation of extruded nuclei from cRBCs SCALING UP- using cord blood CD34 +  cells in bioreactors

20. SOLVENT-DETERGENT FFP The solvent/detergent treatment is an established virus inactivation technology that has been industrially applied for manufacturing plasma derived medicinal products for almost 30 years. Solvent/detergent plasma is a pharmaceutical product with standardised content of clotting factors, devoid of antibodies implicated in transfusion-related acute lung injury pathogenesis, and with a very high level of decontamination from transfusion-transmissible infectious agents.  The solvent/detergent (S/D) pathogen inactivation method was developed in the early eighties for the inactivation of enveloped viruses in plasma-derived medicinal products

21. METHOD 1. Filtration with a 1-μm filter to remove cells and debris 2. FFP is thawed rapidly and treated for 4 h with TNBP ( tri- nitrobutylphosphate ) solvent and with Triton X-100 detergent , both at 1 % 3. TNBP is then removed by extraction with castor (or soybean) oil and the Triton X-100 by hydrophobic chromatography 4. These processes are followed by sterile filtration with a 0.2-μm filter and aseptic packaging into the final product 

22. advantages OF SOLVENT-DETERGENT TREATMENT OF FFPs

23. 1. Viral and bacterial decontamination D econtamination of S/D plasma is guaranteed I mmunological neutralisation D ouble filtration removes cells, cell fragments, and bacteria H ighly effective in disrupting lipid membranes (lipid envelop viruses) Extensive post-marketing clinical experience have clearly shown that the S/D treatment achieves a rapid, irreversible, and thorough inactivation of enveloped viruses Although bacteria rarely contaminate FFP because of its storage conditions, it inactivates some gram-positive bacteria

24. 2. Standardisation & dilution effect The dilution and neutralisation of antibodies and allergens during the industrial process of S/D plasma pooling can reduce the incidence of allergic reactions in recipients Neither anti-human leucocyte antigen (HLA) nor anti-human neutrophil antigen (HNA) antibodies are detectable in S/D plasma as they are diluted and neutralised by the presence of leukocytes or their fragments, which are then removed by the S/D treatment - For this reason countries using S/D FFP have not reported any transfusion-related acute lung injury ( TRALI ) case due to the transfusion of plasma

25. 3. Clinical experience  The high level of safety towards allergic reactions, TRALI and risk of transmission of enveloped viruses and of final standardization of coagulation factor content has recently led to the wide use of S/D plasma A number of clinical studies have analysed in the last two decades the efficacy and safety of this biological agent in various clinical settings, including congenital coagulation deficiencies, acquired coagulopathy of liver disease, coumarin-reversal and surgery 

26. REFERENCES Biopreservation of red blood cells – the struggle with hemoglobin oxidation- The FEBS Journal- VOL 277 Issue 2 Biopreservation of Red Blood Cells: Past, Present, and Future Transfusion Medicine Reviews Volume 19, Issue 2 DJHS Transfusion Medicine Technical Manual Volume 3 Concise Review: Production of Cultured Red Blood Cells from Stem Cells by Eric E. Bouhassira - Stem cell translational medicine Solvent/detergent plasma: pharmaceutical characteristics and clinical experience

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