>100
C/min) and storage at temperatures below À150
C, normally achieved by placing the RBCs in or above liquid nitrogen.
Using HGM, RBCs can be frozen slowly (i.e., 1–3
C/min). With
this method, RBCs are frozen with a final concentration of approximately 40% glycerol (w/v) and stored at temperatures between
65
C and 80
C. Overall, RBC preservation can be extended to at
least 30 years if the correct storage temperature is guaranteed
[12, 13].
Despite their higher costs [14, 15], frozen RBCs have several
advantages over standard RBC products that are mainly related to
the necessary washing procedure, eliminating cell debris, WBCs,
cytokines, and free Hb [16, 17]. Besides cost, the actual use of
frozen RBCs has been hampered mainly by the processing time and
a 24-h outdating period due to potential bacterial contamination if
thawing and washing are performed in a non-closed system
[12]. With the introduction of an automated cell processing system
(ACP215, Haemonetics Braintree, MA), it became possible to
perform both glycerolization and deglycerolization of RBCs in a
functionally closed system, resulting in reduced potential for bacterial contamination and allowing prolonged post-thaw storage
[18, 19]. The ACP215 uses disposable single-use kits having inline
0.22 μm bacteria barrier filters to deliver all fluids needed for the
glycerolization and deglycerolization procedure. Using a sterile
connection device for all tubing connections, a functionally closed
system can be maintained. Using the ACP215 for processing, postthaw storage time of HGM frozen RBCs can be extended to
2–7 days [20] when stored in saline-adenine-glucose-mannitol
(SAG-M) and to 14 [19] or even 28 days [21] when stored in
additive solution-3 (AS-3). The thawed ACP215-deglycerolized
cold-stored product has been shown to be effective in the treatment
of (military) trauma patients and shows superior tissue oxygenation
compared to standard RBC [22–26].
In this chapter, the cryopreservation of red blood cells according to the high-glycerol method using a functionally closed processing system will be described. The described process is used in our
institute for the preservation of rare RBC units. Although the
ACP215 has been developed for processing RBCs according to
the HGM, with some adjustment in the procedure, it can also be
used for deglycerolization of LGM units [27]. Since with the LGM
method, freezing was performed in aluminum containers that cannot be sterilely connected, post-thaw storage of these units is
limited to 24 h.
524
Johan W. Lagerberg
C/min) and storage at temperatures below À150
C, normally achieved by placing the RBCs in or above liquid nitrogen.
Using HGM, RBCs can be frozen slowly (i.e., 1–3
C/min). With
this method, RBCs are frozen with a final concentration of approximately 40% glycerol (w/v) and stored at temperatures between
65
C and 80
C. Overall, RBC preservation can be extended to at
least 30 years if the correct storage temperature is guaranteed
[12, 13].
Despite their higher costs [14, 15], frozen RBCs have several
advantages over standard RBC products that are mainly related to
the necessary washing procedure, eliminating cell debris, WBCs,
cytokines, and free Hb [16, 17]. Besides cost, the actual use of
frozen RBCs has been hampered mainly by the processing time and
a 24-h outdating period due to potential bacterial contamination if
thawing and washing are performed in a non-closed system
[12]. With the introduction of an automated cell processing system
(ACP215, Haemonetics Braintree, MA), it became possible to
perform both glycerolization and deglycerolization of RBCs in a
functionally closed system, resulting in reduced potential for bacterial contamination and allowing prolonged post-thaw storage
[18, 19]. The ACP215 uses disposable single-use kits having inline
0.22 μm bacteria barrier filters to deliver all fluids needed for the
glycerolization and deglycerolization procedure. Using a sterile
connection device for all tubing connections, a functionally closed
system can be maintained. Using the ACP215 for processing, postthaw storage time of HGM frozen RBCs can be extended to
2–7 days [20] when stored in saline-adenine-glucose-mannitol
(SAG-M) and to 14 [19] or even 28 days [21] when stored in
additive solution-3 (AS-3). The thawed ACP215-deglycerolized
cold-stored product has been shown to be effective in the treatment
of (military) trauma patients and shows superior tissue oxygenation
compared to standard RBC [22–26].
In this chapter, the cryopreservation of red blood cells according to the high-glycerol method using a functionally closed processing system will be described. The described process is used in our
institute for the preservation of rare RBC units. Although the
ACP215 has been developed for processing RBCs according to
the HGM, with some adjustment in the procedure, it can also be
used for deglycerolization of LGM units [27]. Since with the LGM
method, freezing was performed in aluminum containers that cannot be sterilely connected, post-thaw storage of these units is
limited to 24 h.
524
Johan W. Lagerberg