Sanne de Bruin

196 Chapter 7 4.6. Prepare 100 ml of 10% ACD-A in 90% PAS-E in a sterile, labeled bag. 4.7. Prepare 11 ml of ACD-A in a sterile, labeled bag. 4.8. Maximal 30 minutes prior to the biotinylation procedure, thaw the stored 50 mg/L biotin-PBS solution, and mix 1 part biotin-PBS-solution to 9 parts PAS-E, to obtain a 5 mg/L biotin-PBS-PAS-E-solution. Fill out 100 ml in a sterile, labeled, bag. 5.1. Obtain the proportion of 50 ml platelet concentrate. 5.2. Transfer 52 grams (50ml) of platelet concentrate to a sterile, labeled bag, using a sterile docking device. 6.0 Washing steps Note: Before every centrifugation step, add a total of 10% ACD-A, to prevent thrombus formation due to centrifugation. 6.1. Dock the bag containing 50 ml of platelet concentrate to the 150 ml 15%ACD- A/85% PAS-E-solution bag. Add this 15%-ACD-A solution to the platelet concentrate. 6.2. Centrifuge the bags at 1700 x g for 10 minutes. 6.3. Remove the supernatant by using the empty bag, discard this bag. 6.4. Dock the bag containing the platelet pallet to the 100ml 10%ACD-A/90%PAS-E bag. Resuspend the platelet pallet by gentle manipulation. 7.0 Incubation with biotin 7.1. Dock the 5 mg/L biotin-PBS-PAS-E-solution to the platelet pellet. Resuspend the platelet pellet by gentle manipulation. 7.2. Incubate for 30 minutes, at room temperature, under gently agitation. 8.0 Washing step and resuspending to original volume 8.1. Add the bag containing 12 ml of ACD-A to the biotinylated platelets. 8.2. Centrifuge at 1700 x g for 10 minutes. 8.3. Remove the supernatant by using the empty bag, discard this bag. 8.4. Dock PAS-E to the platelet pallet, bring to original volume (50 ml). 8.5. Resuspend the platelets pallet by gently manipulation. 8.9. Store the platelets under gentile agitation, at room temperature until use.