atio).
b.Attach the 30 cc syringe with the 25 mL thawing solution to the female luer lock of the Cell Wash/Infusion Bag Set.
c.Open PC-1, PC-2 and PC-3 (see Figure 4 above) and slowly introduce half (~12.5 mL) of the thawing solution to the 25 mL product in the freezing bag while mixing the fluids in the bag using an orbital rotator.
d.Rinse well to remove cells from the bag’s ports.
e.Close PC-3. Open PC-4 and drain the contents from the freezing bag into the transplant bag.
f.Close PC-1 and PC-2. Open PC-3.
g.Slowly add the remaining thawing solution (~12.5 mL) to the transplant bag while mixing the fluids in the bag.
h.Close PC-3.
i.Allow approx. 5 minutes for equilibration.
j.Open PC-1 and PC-2. Pass the diluted HEMACORD back and forth between the transplant bag and the freezing bag in order to more completely wash all cells out of the freezing bag and into the transplant bag.
k.Close PC-1 and PC-2.
l.Attach a syringe with 60 mL thawing solution to the luer lock.
m.Open PC-3.
n.Transfer the 60 mL solution to the diluted HEMACORD in the transplant bag while mixing the fluids in the bag.
o.Repeat with a second 60 mL syringe. The final volume should be approx.170 mL (50 mL diluted HEMACORD with 120 mL thawing solution).
p.Close PC-3. Open PC-1 and PC-2.
q.Pass the reconstituted HEMACORD back and forth between the transplant bag and the freezing bag in order to wash all cells completely out of the freezing bag and into the transplant bag.
r.Close PC-4.
s.Seal the Cell Wash/Infusion Bag Set tubing between PC-4 and IP-1.
t.Cut through seal to separate the transplant bag from the freezing bag.
u.Discard the freezing bag, the luer lock, and the connecting tubing.
v.The reconstituted product can be used for infusion into a patient with or without the additional step of DMSO removal (Section 3.5 below).
w.The recommended expiration time of the reconstituted unwashed HEMACORD is four hours either at room temperature or at 4° C from the time of thaw.
x.Remove a small volume from the reconstituted product for Complete Blood Counts (CBC), CFU, CD34+ counts, viability, and sterility samples (bacterial and fungal cultures) as per transplant center procedures.
NOTE: If more than four hours elapse between thawing and infusion, an aliquot of the product should be removed and tested immediately before administration to the patient to determine the cell viability of the infused product.
y.Call the Transplant Unit to advise them that the product is ready for infusion if you do not intend to remove the cryoprotectant.
3.5 Removing the Cryoprotectant (Washing)
a.Plac the transplant bag and the transfer bag in a centrifuge cup.
b.Fully support the transplant bag with inserts to prevent formation of creases during centrifugation (as shown in Figure 7 below).
Figure 7.
c.Close SC-1 securely.
d.Centrifuge at 400 x G for 20 minutes at 10°C.
e.After centrifugation, carefully remove the bags from the centrifuge bucket without disturbing the cellular pellet in the transplant bag.
f.Place the transplant bag in the plasma extractor.
g.Using SC-1 to adjust the flow, very slowly transfer approximately 2/3 of the supernatant (Supernatant-1) to the transfer bag avoiding the passage of cells.
h.Leave approximately 1/3 of supernatant with the cells (white and sedimented red cells in the diagram above). If you detect passage of cells to the transfer bag, return the contents to the transplant bag, resuspend the cells, an |
