3. Gently remove the unit from the centrifuge and place it in a
plasma extractor.
4. Squeeze weld and express all air and visible supernatant glycerol
to the transfer bag.
5. Place a Kocher clamp on the tubing between the two bags.
6. Remove the glycerolized RBC from the plasma extractor and
thoroughly mix the unit.
3.1.5 Freezing the Unit
The glycerolized cells should be frozen slowly, with a cooling rate
of 1–3
C/min. This can be achieved by placing the glycerolized
units in a cardboard box at the bottom of a chest freezer:
1. Sterilely dock a 150 mL transfer bag to the freezing bag.
Squeeze weld and transfer about 10 mL of cell suspension to
the transfer bag. This will be used for quality control and filling
reference samples (cryovials for blood group typing).
2. Weigh the glycerolized unit and record the net weight of
the unit.
3. Label the unit and freezing box as required.
4. Fold the unit in half with the ports and tubing tucked under
the top of the bag. Place the unit inside a sealable bag.
5. Seal the plastic bag using an impulse sealer so that there is as
little trapped air as possible, preferably by using a vacuum
sealer.
6. Place the bag plus the reference samples (cryovials containing
1.5 mL glycerolized RBC solution) inside a cardboard
freezing box.
7. Place the cardboard box in a À80
C freezer. Units should be
frozen in a location within the freezer where optimal freezing
can occur (preferably at the bottom of an empty chest freezer).
To avoid improper freezing, the units should not be stacked on
each other. Optimal freezing rate is 1–3
C/min for the HGM.
8. After 24 h, the units can be stacked and stored in the chest or
upright À80
C freezers.
3.2 Thawing
and Deglycerolization
of Red Blood Cell
Concentrates
In the deglycerolization process, the ACP215 first dilutes the glycerolized RBCs with 50 mL of 12% NaCl while shaking the unit
thoroughly on the shaking platform. After an equilibration delay,
340 mL of 0.9% NaCl/0.2% glucose solution is added while using
the shaking platform to ensure adequate mixing. The diluted cells
are transferred to the bowl where the supernatant glycerol and
saline are removed. The product is pumped back to the bag, and
400 mL of 0.9% NaCl/0.2% glucose solution is added while using
the shaking platform to ensure adequate mixing. In both dilution
steps, the dilution rate is calculated by the ACP to prevent an
Cryopreservation of Red Blood Cells
529
plasma extractor.
4. Squeeze weld and express all air and visible supernatant glycerol
to the transfer bag.
5. Place a Kocher clamp on the tubing between the two bags.
6. Remove the glycerolized RBC from the plasma extractor and
thoroughly mix the unit.
3.1.5 Freezing the Unit
The glycerolized cells should be frozen slowly, with a cooling rate
of 1–3
C/min. This can be achieved by placing the glycerolized
units in a cardboard box at the bottom of a chest freezer:
1. Sterilely dock a 150 mL transfer bag to the freezing bag.
Squeeze weld and transfer about 10 mL of cell suspension to
the transfer bag. This will be used for quality control and filling
reference samples (cryovials for blood group typing).
2. Weigh the glycerolized unit and record the net weight of
the unit.
3. Label the unit and freezing box as required.
4. Fold the unit in half with the ports and tubing tucked under
the top of the bag. Place the unit inside a sealable bag.
5. Seal the plastic bag using an impulse sealer so that there is as
little trapped air as possible, preferably by using a vacuum
sealer.
6. Place the bag plus the reference samples (cryovials containing
1.5 mL glycerolized RBC solution) inside a cardboard
freezing box.
7. Place the cardboard box in a À80
C freezer. Units should be
frozen in a location within the freezer where optimal freezing
can occur (preferably at the bottom of an empty chest freezer).
To avoid improper freezing, the units should not be stacked on
each other. Optimal freezing rate is 1–3
C/min for the HGM.
8. After 24 h, the units can be stacked and stored in the chest or
upright À80
C freezers.
3.2 Thawing
and Deglycerolization
of Red Blood Cell
Concentrates
In the deglycerolization process, the ACP215 first dilutes the glycerolized RBCs with 50 mL of 12% NaCl while shaking the unit
thoroughly on the shaking platform. After an equilibration delay,
340 mL of 0.9% NaCl/0.2% glucose solution is added while using
the shaking platform to ensure adequate mixing. The diluted cells
are transferred to the bowl where the supernatant glycerol and
saline are removed. The product is pumped back to the bag, and
400 mL of 0.9% NaCl/0.2% glucose solution is added while using
the shaking platform to ensure adequate mixing. In both dilution
steps, the dilution rate is calculated by the ACP to prevent an
Cryopreservation of Red Blood Cells
529