Date on Senior Honors Thesis

5-2023

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Biology

Author's Keywords

Lyophilization of Red Blood Cells; Allosteric Regulation of Oxygen Binding; Freeze-Dried Red Blood Cells (RBCs); Freeze Drying; Blood Oxygen Binding System (BOBs); Hemoglobin Oxidation

Abstract

Blood transfusion is the single most often performed lifesaving procedure in hospitals worldwide. Unfortunately, packed red blood cells (RBCs) used for transfusion can only be stored for 42 days at 4 °C before being discarded due to irreversible damage that occurs during storage. Any reduction in available RBCs for an extended period can lead to blood shortages. To increase the shelf-life of RBCs, we investigated freeze-drying (lyophilizing) in the presence of the non-toxic sugar trehalose as a method for long-term preservation. However, the oxidative stress of the lyophilization and storage processes can compromise the functionality of these cells, and the oxygen-transport protein hemoglobin may convert via oxidation into the nonfunctional methemoglobin (MetHb) form. Therefore, it is necessary to quantify the percentage of MetHb after rehydration to determine the functionality of lyophilized RBCs. Likewise, investigating the allosteric regulation of oxygen binding by hemoglobin is required to establish the suitability of the rehydrated product for transfusion. We found that prevention of oxidation of hemoglobin to methemoglobin during freeze-drying is challenging, and methemoglobin concentration increased during storage of the desiccated RBCs. This increase occurred during storage at high and low humidity and under vacuum. However, RBCs fortified with ascorbic acid before lyophilization and stored at 0% relative humidity showed the lowest methemoglobin content. Moreover, our data demonstrate that allosteric regulation of hemoglobin is maintained after lyophilization and rehydration. Optimizations of the freeze-drying and storage processes are being performed to develop this technology further.

Lay Summary

When you go to the grocery store there are two types of milk that you can buy, bottled liquid milk and powdered dry milk. Bottled milk is in the fridge, must be kept at low temperature and it last no longer than few months before it expires. Dry milk on the other hand, is on the shelf, it can be stored at room temperature, and it last for years – all you need is to rehydrate it with water. Currently blood is more like liquid milk, it must be stored at 4 oC and can only last for 42 days before it should be discarded. Therefore, complex infrastructure and continuous blood supply must be maintained to avoid blood shortages. This is extremely critical since blood transfusions are the most performed lifesaving procedure – the only thing that can save a patient who has lost blood is blood. Drying blood to a powder form is a potential way to solve this issue, since it will eliminate the need for refrigeration and extend the shelf life of the blood thus buffering against any distribution in the blood supply.

The purpose of our research is to develop blood powder and to make sure that this form of blood will function just as well as liquid blood after rehydration and that it is suitable for transfusion. The research focuses on the function of hemoglobin – the protein responsible for binding oxygen and transporting it throughout the body. The research's goal is to ensure that the hemoglobin of powder blood can bind oxygen in the same way as liquid blood.

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