Freezing of Red Blood Cells Admendments and Questions

Hey guys, thanks for the questions.

I have some amendments to my first post. First of all, not all the blood that is going to be frozen has to be quarantined. The blood that I was dealing with was malaria blood. Hence that is why they froze it. The donor may come from malaria countries and that may want to do a retest in the future. Hence they decided to freeze it while pending tests in the future. Normal red blood cell that you are going to freeze does not have to be quarantine. The computer system will record the freezing process once you do transformation. Secondly, the blood group has actually been written on the bag. What is missing is the blood group in the recipient card. So you have to actually record the blood group on the recipient card, as the card will be kept somewhere else. Thirdly, I was confused about the plasma extractor. I thought that it was a specific term that was used only when you want to extract plasma. After I was posted to the freezing lab, I was next posted to the centrifuge bay lab. In the centrifuge bay lab, the plasma will be removed from the whole blood and additive solution called SAGM (saline-adenine-glucose-mannitol) has been added to the packed cells. So when you are doing freezing, what am I actually squeezing out is the SAGM and the buffy coat. Fourthly, I would like to emphasize that the HCT has to be between 70 to 80 percent to pass. And lastly, during the addition of glycerolyte solution, the first pause is when you added 50ml for 5 mins and second pause is for another 50ml for 2 mins. When you done the second spin, you have to squeeze out 320 ml of glycerolyte solution out of the primary bag into the transfer bag. Lastly, after the addition of glycerolyte, the freezer bag is left standing for about 15 mins. This is to allow more time for the glycerol to replace the water inside the cells. I am truly sorry for the mistakes that I did in my earlier post. I doubled check what I did with my trainer and he pointed out some of my mistakes and misconceptions.

And now for the questions…

Hi Indah,
After adding the 100ml of the glycerolyte solution, why must the flow be stop? is it not possible to add the total volume at one go? also, what is the purpose of the shaker? Thanks!

zi shuang
0703383J

Thanks Zi Shuang for the question. The shaker is to mix the red cells with the glycerolyte solution as the solution flows into the freezer bag. The faster the speed of the shaker, the faster the flow of the solution out of the Glycerolyte 57 Solution bottle and into the freezer bag. It should be added in a stepwise fashion. So not too fast shaking. The pauses are to allow time for gradual equilibration between the red cells and the glycerol. Hence time is given for the glycerol to enter the cells and replace the water that is present in the red cells.

Hi Indah,

What is the purpose of the Glycerolyte 57 solution?

Qingling
0703433C

Thanks Qing Ling for the question. This solution is basically glycerol. It is a cryoprotective agent. During freezing, the water present in the cells can become ice crystals and cause injury to the cell membrane. This can lead to cell lysis. The glycerol is able to protect the cell from freezing injury and hence enhance cell recovery during thawing.

Hi Indah,
May I know what will happen to the blood that does not complete the freezing process within 4 hours?

Liyana
0703827F

Thanks Liyana for the question. The 4-hour rule is incorporated based on the requirement of the American Association of Blood Banks (AABB). Packed cells and whole blood should not be left out in the room temperature for very long as it must be maintained at temperature of 2-6°C. I am not too sure what are the procedures if you cannot complete the freezing process within 4 hours. One lab technician can only handle 6 blood bags at a time. There is only one centrifuge in which 6 blood bags can fit. Haha. So should be okay.

Hey Indah!
very detailed explanation. although i tink if you have some photos would be better? but i can imagine the primary and the freezer bag together and all that. haha.
ok. question:

1) after transferring the blood to the freezer bag, how come there's a need to also centrifuge the primary bag as well since it has to be discarded eventually?

2)i was just wondering, since the blood are all frozen, when the blood is needed, what is done to de-froze (like the procedures) especially in the case of emergency?

thank you indah!

Janice Yeh :) Grp 6
0701885F

Thanks Janice for the questions. For the 1st question, you are referring to the 2nd spin. The primary bag is not discarded as afterwards you have to transfer 320ml of the glycerol (top layer) out of the freezer bag. For the 2nd question, the process is thawing and deglycerolisation. I did not get a chance to view this as I was practicing on the freezing part. I have only 2 days in this lab before being rotated. so ya. Perhaps, if I have sometime during my major project, I can request to view the process and put it as one of my post.

Indah!
Come up and join me for lunch someday okay.
For the blood, we're only getting the packed cells frozen right?
And erm. This process is quite lengthy and wouldn't be cheap i guess. So, what are the blood types that you guys will freeze? Is it by patient's request? Or just some rare blood group?

Poh Yan Hong
0703979E / TG01

Hey babe… thanks for the question. I don’t have the time to eat with you guys cos the eating place is far away. I made my mom to cook food for me early in the morning. Haha. And I eat with the rest of the staff there at the pantry. But if I managed to get out early for lunch then I will msg you.
Ok, back to the question. Lengthy, yes. Cheap, I don’t know. I guess you just need to buy boxes of glycerolyte solution bottles and transfer bags. About the types of blood that we froze, im not too sure if there are any preference. I guess if we are to stockpile blood, then blood group O is very popular as it is the second choice for transfusion of packed cells as it is universal donor and can be used as emergency blood. I think we also freeze rare blood groups such as Bombay blood.

Hi Indah, :)

I'm just curious why do we need to place the bag into the water bath for 30 mins and at 37 degree C? What is the bag and its purpose?

Rachel Gan :)

Thanks Rachel for the question. The bag is the primary bag. If you are referring to the type of bag, I think it is the Baxter bag, 450ml. There are other types of bag as well such as Terumo bag. The purpose of placing the bag in the water bath before adding the glycerol is to let the cells warm up and the membrane more fluid so that later it is easier for the glycerol to enter the cells and the water to go out of the cells. The timing is important. We have a stopwatch alarm to time for exactly 30 mins.

Hello Indah,

Hope you are well =)

Are you meaning to say that the process involves removing the plasma and the buffy coat ? Also, why must some plasma be added back into the primary bag until the weight is increased by 25g ?

Thanks,

Ng Tze Yang Justin
0703747F

Hey Justin. I’m very well. I apologise for the plasma part. It is actually SAGM – an additive solution you added to the packed cells as explained in my amendments. Why you add back 25 g? I was told that it is method for the HCT testing. A failed HCT is retested again. A repeated HCT failure means you have to do the entire process again. The HCT may be too low indicating your cell recovery during thawing will likely to be low. I’m guessing that too high HCT could be due to centrifuging effects where the cells are really packed together. To get an accurate HCT as possible, it is best to add back 25g.