WEEK NUMBER THIRTEEN

HELLO EVERYONE!!!!
Hope you guys are doing well.

This week, I will be touching on a topic which may be foreign to some of you. It will be on glycerolization/freezing and deglycerolization/thawing of red blood cells. This process is done in the cryopreservation lab of the blood bank.

Glycerolization/Freezing
Freezing RBCs with glycerol dates back to the 1950s. Frozen RBCs can be stored for up to 10 years for:
1. Patients with rare phenotypes
2. Autologous use
3. In case of national emergencies in which blood cannot be dispositioned out to hospitals quickly enough to prevent expiration.

The resulting deglycerolized product is free of leucocytes, platelets, and plasma due to washing process. Cryoprotective agents can be categorized as penetrating and nonpenetrating. A penetrating agent involves small molecules that cross the cell membrane into the cytoplasm. The osmotic force of the agent prevents water from migrating outward as extracellular ice is formed, preventing intracellular dehydration. An example of a penetrating agent is glycerol. An example of a non-penetrating agent is hydroxyethyl starch (HES). This comprises large molecules that do not enter the cell but instead form a shell around the cell, preventing loss of water and subsequent dehydration.

Two procedures used for glycerolizing RBCs are high glycerol and low glycerol methods. The methods differ in the equipment used, the temperature storage and the rate of freezing. Most blood centres practice the high glycerol method.

High Glycerol (40% w/v)
This method increases the cryoprotective power of the glycerol, thus allowing a slow, uncontrolled freezing process. The freezer is generally a mechanical freezer that provides storage at -80oC. This particular procedure is probably the most widely used because the equipment is fairly simple and the products require less delicate handling. It does however require a large volume of wash solution for deglycerolization. RBCs are frozen within 6 days of collection when the preservative is CPD or CPDA-1 and up to 42 days when preserved in AS-1, AS-3 and AS-5. AABB Standards states RBCs must be placed in the freezer within 4 hours of opening the system. It is advisable to freeze a sample of donor serum in the event additional testing is required for donor screening.

Low Glycerol (20% w/v)
In this method, the cryoprotection of the glycerol is minimal, and very rapid, more controlled freezing procedure is required. Liquid nitrogen (N2) is routinely used for this method. The frozen units must be stored at about -120oC, which is the temperature of liquid N2 vapour. Because of the minimal amount of protection by the glycerol, temperature fluctuations during storage can cause RBC destruction.

Deglycerolization/Thawing
Red cells that have been frozen require thawing and deglycerolization before they can be safely transfused. Glycerol will be removed from RBC to avoid in vivo and/or in vitro heamolysis.

The thawing process takes approximately 30 minutes and involves immersion of units into a 37oC water bath and washing the RBCs with solutions of decreasing osmolarity (eg. 12% NaCl, 1.6% NaCl, 0.9% NaCl, + 0.2% dextrose). An exception to this rule is a donor with sickle cell trait in which RBCs would haemolyze upon suspension in hypertonic solutions; in this case the cells would be washed in 12% NaCl and then 0.9% NaCl with 0.2% dextrose, omitting the 1.6% solution. Automated continuous-flow instruments can be utilized for washing. In our blood bank, a machine called the Haemonetic 215 is used. Once the RBCs have been deglycerolized, the unit is considered an open system with an expiration date of 24hrs and is stored at 1oC to 6oC.

Hope my post this week has enlightened you on some of the processes that occurs in a blood bank. Till next time..... Adios!!!

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