Background

Red blood cell (RBC) units are administered routinely into patients expressing a wide range of acute and chronic conditions (e.g., anemia, traumatic bleeding, chronic diseases, and surgery). The modern blood banking system has been designed to answer this need and assure a continuous, high quality blood supply to patients. However, RBCs units can be stored under hypothermic conditions for only up to 42 days, which leads to periodic shortages. Cryopreservation can solve these shortages, but current freezing methods employ high glycerol concentrations, which need to be removed and the cells washed prior to transfusion, resulting in a long (more than 1 hour) and cumbersome washing step. Thus, frozen RBCs have limited use in acute and trauma situations. In addition, transportation of frozen samples is complicated and costly. In this context one recognized and attractive alternative to long-term storage of RBCs by freezing is to freeze-dry (lyophilize) them, yielding a dry RBC product (freeze-dried RBCs). It is expected that freeze-dried RBCs will be a product that can be stored for extended periods without refrigeration or degradation, eliminating expiration concerns.

Methodology

Confidential military project to develop a process for freeze drying red blood cells (RBC) and demonstrate the function of the cells in an animal model. Research was aimed at testing the hypothesis that the stabilization of RBC membranes by sugars, cholesterol or antioxidants, with the incorporation of three new technologies (MTG, RFV, DS) into the lyophilization process will generate homogenous physical conditions throughout a volumetric sample during the different steps of the lyophilization process, and in combination with the available knowledge base on stabilizing solution, allow a significant increase in the number and quality of red blood cells that can survive the lyophilization process to a level that will allow clinical transfusion.

Principal findings

Using a special stabilizing solution in combination with a directional freezing devices and support equipment, results consistently indicated ability to prepare 500-mL frozen RBC products (containing 250 mL of packed RBCs and 250 mL of IMT-1) that, after thawing, exhibit excellent characteristics. The thawed products have more than 98% recovery of RBCs, with 1% to 5% free hemoglobin (Hb). The recovered RBCs respond normally to hyperosmotic stress (osmotic fragility), high ATP, and 2,3- DPG levels, and normal oxygen association/dissociation curves. In addition, there was no difference in RBC post-thaw deformability compared with deformability before freezing. Studies with the recovered cells in large-animal models showed that more than 80% of the frozen-thawed RBCs were circulating 24 hours post-transfusion.

Preliminary experiments that were performed on small-volume samples (1 mL) and with a low hematocrit (5%) showed that it is possible to recover freeze-dried RBCs with good morphology and integrity, albeit in relatively low yield. After lyophilization and rehydration, 50% of the cells were recovered and showed normal morphology. ATP and 2,3-DPG levels post-rehydration were similar to the values of fresh blood (2,3-DPG prior freezing that showed 7.6 ± 0.1 and post-rehydration results were 6.2 ± 1; ATP values prior freezing were 7 ± 0.3 umol/grHb and after rehydration values 2.7 ± 1.5 umol/grHb, respectively). Finally, the association dissociation curve showed good oxygen uptake of the rehydrated blood. It should be noted that even if produced on a small scale, freeze-dried RBCs would be useful as long-lived reagents for routine use and as identification samples carried by each individual for rapid cross-matching and testing.

Conclusions

Lyophilization of red blood cells would allow storage not only in hospitals (permanent structures and mobile), but could also be generated as autologous units for, and carried by, select military personnel. Moreover, such a product would also serve rural areas and under-developed countries where limits in electricity and transportation prevent the appropriate and timely usage of RBC transfusions. Finally, freeze-dried RBCs have the potential to meet the need for the consistent supply of a safe and sterile worldwide blood supply, eliminating the need for frozen storage and assuring significantly longer shelf life for both blood and RBC-based reagents.

Source

Freeze-Drying of Red Blood Cells: The Use of Directional Freezing and a New Radio Frequency Lyophilization Device
A. Arav, Y. Natan
Biopresevation and Biobanking, vol. 10, no. 4 (2012)
For more information click here.

Freeze-Drying of Red Blood Cells
A. Arav, Y. Natan
The Journal of Trauma® Injury, Infection, and Critical Care, vol. 70, no. 5, (2011)
For more information click here

Supporting information

Figure 1. Scanning electron microscope images of freeze dried red blood cells
Left – dehydrated cells; Right – normal appearance

Figure 2. The effect of cell concentration on survival of lyophilized RBCs

Figure 3. The effect of DMSO concentration on post rehydration of RBCs viability

Figure 4. Assessing different rehydration solutions

Figure 5. The effect of storage temperature

Fresh Hypothermia
(5-21 days)
Frozen Freeze dried
Biochemistry
pH 7.10±0.1 6.3±0.1 7.04±0.09 N/A
2,3 DPG 11.5±6.2 0.4±0.2 4.11±0.4 6.2±0.9
ATP 5.9±0.4 1.8±0.3 4.76±0.42 2.7±1.5
Free Hgb <1% <4% 2.54±0.33 20%-50%
Membrane function
Osmotic Fragility Comparable to fresh Comparable to fresh Right shift – cells survive better at hyper osmotic solution
Membrane deformability Gradually deteriorates Comparable to fresh N/A
Typing Preserved Major+Minor Preserved Major+Minor Preserved Major+Minor
Cell function
O2 association dissociation Altered due to changes in ph and 2,3 DPG Comparable to fresh N/A
In vivo
Cell survival in circulation 90% after 24 hours N/A >80% after 24 hours (dog)
60%-80% after 24H (donkey)
40% after 2 hours
(donkey)

Table 1. Red blood cell quality comparison