Freezing of Red Blood Cells

Hi. I’m Indah and I’m attached to a blood bank laboratory section.

There are many different laboratories and I was rotated from one laboratory to another. One of the laboratory sections that I was posted to is the freezing of blood. Blood can be frozen and stored for up to 10 years. This can be applied in situations like rare blood groups or autologous blood for future surgery. I am going to bring you through the process that I have done.

Blood that is going to be frozen has to be quarantined in the blood bank computer system and it will not be allowed for issue to the hospitals for transfusion. For every blood and its derived blood components that are quarantined, a unique quarantine number will be issued and is recorded in the quarantine logbook. The blood will be stored in the pending cold room. On the day that the blood will be frozen, it will be transfer to a refrigerator in the freezing laboratory, where it will be stored temporarily at 2 to 8°C. Blood that will have to be frozen are usually closed to its expiry date and freezing has to be done before expiration. I did freezing on red blood cells. The entire freezing process will have to done within 4 hours.

First of all, write down the blood group on the bag that will be frozen. If it is a single bag, the blood will need to be joined to a transfer bag to become a double bag. This can be done using a connector machine that will connect the two bags via the middle tubing. Tie a knot on the connected tubing to stop blood from flowing from the primary bag to the transfer bag. The double bag is then fold to fit in a centrifuge bucket for centrifugation. This is known as the first spin at 3200rpm for 4 minutes at 20°C. While waiting, we will do some documentation such as the recording the date of freezing, the blood donation number from the bag, the blood group, the quarantine number and the lot number of the Glycerolyte 57 solution bottle used.

After the first spin, carefully remove the blood bags from the centrifuge and place the primary bag on a plasma extractor. Untie the knot in the tubing and the plasma and the buffy coat (i.e. the top layer) will be squeezed out into the transfer bag. Then, while stopping the flow of blood from the two bags using your fingers, place the primary bag on a weighing scale. Fold the transfer bag in such a way that the buffy coat will settle to the bottom and add some plasma back into the primary bag until the weight is increased by 25g. Seal the tubing in between the two bags and dispose the transfer bag. Next, strip the tubing of the primary bag, ensuring no bubbles. Seal to form a segment and remove the segment from the rest of the tubing. Paste a donation number label on a test tube. Cut the separated segment and pour the blood into the test tube. Do a 1:1 dilution with NaCl. Test for the HCT using the automated ADIVA 120 machine. A pass is above 70% and a report is printed out. After that, weigh the blood again. Make sure to tare first with an empty bag. Record the volume of the blood (Volume = Mass/Density).

Place the bag inside a water bath for 30 minutes at 37°C. In the meantime, prepare the set up for the transfer of blood from the primary bag to the freezer bag (2000ml) and the glycerolyte solution. Connect the tubing from the glycerolyte bottle to the freezer bag but do not allow the flow of glycerolyte to the freezer bag by locking the clips. After 30 minutes, remove the bag from the water bath. Clean the needle of the freezer bag with methylated spirit and poke the needle to connect to the primary bag. Hang the primary bag upside down and allow the blood to drain to the freezer bag. After all the blood has been drained, transfer the whole set up to a shaker. Hang the glycerolyte bottle and unlock the clips, allowing glycerolyte solution to flow into the freezer bag and mix with the blood. After adding 100 ml, pause the flow by locking the clips and stop the shaker for 5 minutes. After adding another 50ml, pause again for 2 minutes. Transfer the rest of the glycerolyte solution, giving a total volume of 400ml. Lock all clips so that blood will not flow out of the freezer bag. Seal the tubing between the bottle and the freezer bag and discard the bottle.

Fold the freezer bag into a plastic bag and, together with the primary bag, they are centrifuged at 2500rpm for 10 minutes at 20°C. This is known as the second spin. Again carefully place the freezer bag onto the plasma extractor and squeeze out 50ml of the glycerolyte solution (i.e. top layer). Seal the tubing and discard the primary bag. Strip the freezer bag tubing.

Transform the blood product in the computer system into frozen red blood cells. (i.e. the transformation is done by a freezing laboratory staff as I do not have access to the computer system). Label the blood card with a frozen red blood cells label. Fold the bag in such a way that the tubing will not overlap with each other and place in a metal plate that is labelled with the donation number, blood group, quarantine number and date of freezing. Place the metal plate in the freezer at –80°C, laying it flat so that the blood will not sink to one side.

Some of the difficulties I faced while doing this were tying a knot, stripping the tubing and transferring the wanted volume from one bag into the other as the weight of the weighing scale keep on fluctuating.

Indah
0705361D