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:

1. Pool the blood from same donor (note blood collected in 63ml anticoagulant).
2. Decanter 50% of blood and aliquot, 1ml per screw top tube. Freeze in dry ice/ ethanol 96 bath. Store at -80^oC.
3. Aliquot remaining blood ~45ml per centrifuge tube, spin at 1800g with acc 4 dec 4 for 20min at 4 ^oC.
4. Decanter off plasma fraction, pool and aliquot, 1ml per screw top tube. Freeze in dry ice/ ethanol 96 bath. Store at -80^oC.
5. 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 -80^oC.
6. 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 -80^oC.
7. vii) Transfer onto dry ice for transport to storage facility at NIBSC (-80 ^oC).