d repeat the centrifugation or centrifuge only the Supernatant-1 bag (as described below).
i.Empty the tubing between the bags by pushing air from the transfer bag to the transplant bag.
j.Close SC-1.
k.Seal the tubing between the bags close to the transplant bag. Cut through the seal and disconnect the transfer bag with the Supernatant-1 from the transplant bag with the cellular pellet (product).
l.Resuspend the cellular pellet by slowly adding (with a syringe) 25-50 mL of the thawing solution through the IP-1, with continuous mixing. The resuspended cells constitute the Sediment-1 (the graft).
m.The weight of the empty transplant bag is 23.6 g if cut and sealed as shown below (Figure 8). Calculate the weight of the Sediment-1 by weighing the filled transplant bag and subtracting 23.6 g.
n.Remove a small volume from the Sediment-1 for cell count, viability determination, and sterility (bacterial and fungal cultures).
o.The recommended expiration time for HEMACORD after the removal of the cryoprotectant is 24 hours from the date and time of thaw. Store the product at 4°C in a blood storage refrigerator until the product is used.
Figure 8.
p.Inspect the supernatant for escaped cells, even if there is no appearance of escape.
q.Express 10 mL from the Supernatant-1 bag into a conical centrifuge tube (accurate volume will help the accuracy of estimations).
r.Centrifuge at 600 x G for 10 minutes.
s.Carefully aspirate 9.5 mL of supernatant without disturbing the (possible) cell pellet in the tip of the tube.
t.Resuspend the cell pellet thoroughly in the 0.5 mL of supernatant and load into a cell-counting chamber.
u.Count the nucleated cells per microliter and calculate the total number of cells in the remaining volume of Supernatant-1.
v.Determine the number of nucleated cells in Supernatant-1 per kg of patient’s weight. The transplant physician may decide whether to add these cells to Sediment-1 cells (the graft) in cases where the Sediment-1 cell dose is low or borderline.
w.If collection of escaped cells from the bag containing Supernatant-1 is desired:
1.Centrifuge the Supernatant-1 bag at 400 X G for 20 minutes at 10°C to sediment the cells.
2.In a laminar flow hood, connect a 300 mL transfer bag to the bag containing the centrifuged product.
3.Position the bag in the plasma extractor and express the new supernatant (Supernatant-2) into the transfer bag, leaving the sedimented cells (Sediment-2) in the original bag.
4.Seal the tubing between the bags, cut through the seal, and disconnect the transfer bag with the Supernatant-2 from the original bag with the Sediment-2.
5.Resuspend the Sediment-2 in 10-15 mL thawing solution, using a syringe and mixing gently. The transplant physician may modify the volume for injection if preferred. If volume modification is desired, resuspend the cellular pellet to the final volume by injecting with thawing solution.
6.Weigh the Supernatant-2 bag and the Sediment-2 bag, and calculate the volumes by subtracting the weight of the empty bags similarly sealed.
7.Remove a small volume from the Sediment-2 for cell count, viability determination, and sterility testing.
x.Bring the transplant bag (Sediment-1 bag) to the Transplant Unit, even if the second bag (Sediment-2 bag) is being prepared; the second bag can be infused separately afterwards.
4. ADMINISTRATIVE REQUIREMENTS
a.Prepare a report on the procedure. Note the condition of the HEMACORD bag, includi |
