Equipment :
Eppendorf EDOS 5222 Liquid dipensing system
Refrigerated Centrifuge capable of spinning at 1800g with
controllable acceleration and deceleration
Baxter -450 ml standard double blood pack / CPDA-1 / PL-146-PL-1240
/ 16 gauge needle code: FGR8211
Baxter- 450 ml OptiPure® RC quadruple blood pack / CPD-SAG M / soft
housing red cell filter / PL-146-PL-1240 / 16 gauge needle Code:
FGR8478B
Solution :
citrate-phosphate-dextrose-adenine-1(CPDA-1) anticoagulant,
supplied with blood bags 63ml
saline, adenine, glucose and mannitol (SAG-M) red cell optimal
additive solution, supplied with blood separation bags 100ml
Procedure:
- Pool the blood from same donor (note blood collected in 63ml
anticoagulant).
- Decanter 50% of blood and aliquot, 1ml per screw top tube.
Freeze in dry ice/ ethanol 96 bath. Store at -80oC.
- Aliquot remaining blood ~45ml per centrifuge tube, spin at
1800g with acc 4 dec 4 for 20min at 4 oC.
- Decanter off plasma fraction, pool and aliquot, 1ml per screw
top tube. Freeze in dry ice/ ethanol 96 bath. Store at
-80oC.
- Decanter off Buffy coat fraction and pool. This may be further
spun (as previously for whole blood to ensure fraction is as pure
as possible). Buffy coat should then be aliquoted, 0.25ml per screw
top tube. Freeze in dry ice/ ethanol 96 bath. Store at
-80oC.
- Decanter off Red Blood Cell fraction and pool. Add SAG-M at a
proportion of 2/9 of the volume of the original whole blood spun
for fractionation (ie volume of SAG-M added =(total volume of blood
with anticoagulant -(63*no of blood packs used))*proportion used
for fractionation *2/9), mix. For example for 513ml of total blood
collected with anticoagulant in one pack, 1/2 used for
fractionation; amount of SAG-M = (513- (63*1))*0.5*(2/9). Aliquot,
1ml per screw top tube. Freeze in dry ice/ ethanol 96 bath. Store
at -80oC.
- vii) Transfer onto dry ice for transport to storage facility at
NIBSC (-80 oC).