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Our lab is a member of the SCARF scheme but we seem to be receiving less and less rare cells and wondered whether all labs are experiencing this?  We currently store rare cells in LN2 but we are being encouraged to reduce our use of LN2 due to the H&S risks.  I would be grateful to hear how other sites freeze and store these cells?

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58 minutes ago, catm said:

Our lab is a member of the SCARF scheme but we seem to be receiving less and less rare cells and wondered whether all labs are experiencing this?  We currently store rare cells in LN2 but we are being encouraged to reduce our use of LN2 due to the H&S risks.  I would be grateful to hear how other sites freeze and store these cells?

The SCARF membership used to be held by NHSBT-Tooting Centre, where Alan Gray used to organise units from our own rare donors (by "ours", I mean the NHSBT, not Tooting alone - although most of them were Tooting donors, because of the rare donor screening programme, run by Alan, which found many rare donors).  Since the latest reorganisation of RCI, I do not know who is holding the membership, but I do know that I was told that NHSBT are sending out fewer rare donor samples.  Your own Head of Laboratory may know or, if not, give Alan a ring at Tooting.  SCARF runs on a reciprocal arrangement, so that, if NHSBT does not send in samples itself, it will not receive samples from others.  In addition, and, as far as I know, since the rare donor screening programme has been removed from Tooting RCI and has gone to Filton RCI, the whole thing has ground to a halt, because of pressure of work - but this information may well now be out of date.

I am simply amazed that you are still using liquid nitrogen to freeze and store your rare red cells, for the very reason you give; H&S.

You should have access to a -80oC freezer, as these were being purchased for all Centres not too long ago (certainly, I was still working when this was offered, and I only retired at the end of October 2016), in which case you can go freezing in glycigel.  There is a recipe for this and directions for use in the library section of this site (I know, because I put it on there, although it was written by Alan) under SOPs.  You should be able to get an up-to-date version from QPulse.  Of course, I have made the assumption here that you are working for NHSBT, whereas you may, of course, be working for the Northern Irish, Scottish or Welsh Blood Services, so, if so, if you telephone 0203 123 8300 and ask to speak to Alan Gray (or Doris Lam, as Alan now works part time), you should be able to obtain an SOP.

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Thanks Malcolm

That is really helpful . I will get in touch with Alan/Doris for further information :D.

 

 

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31 minutes ago, exlimey said:

What, precisely, are the "H&S" risks?

As I understand it, when liquid nitrogen boils, the gaseous nitrogen vapours, which are, of course, invisible, do not mix immediately with the rest of the air.  This nitrogen gas is "heavy", and sinks to the floor, and anyone not aware of the situation can become asphyxiated, as the oxygen level at "breathing height" (for want of a better way of putting it) plunge.  There was a near fatal case of this at the NHSBT National Frozen Blood Bank a few years ago.

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3 hours ago, Malcolm Needs said:

As I understand it, when liquid nitrogen boils, the gaseous nitrogen vapours, which are, of course, invisible, do not mix immediately with the rest of the air.  This nitrogen gas is "heavy", and sinks to the floor, and anyone not aware of the situation can become asphyxiated, as the oxygen level at "breathing height" (for want of a better way of putting it) plunge.  There was a near fatal case of this at the NHSBT National Frozen Blood Bank a few years ago.

I must confess.....I was poking the bear. I have worked with LN2 for 25+ years and personally believe the risk of asphyxiation is highly over exaggerated.  Any room with adequate ventilation (and that's a very loose term) is safe. Most interactions with an LN2 environment are short term, except perhaps for the worker(s) at the NHSBT National Frozen Blood Bank where special ventilation systems should have been designed into their operation.

I was expecting answers involving cryogenic burns, too.

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1 hour ago, exlimey said:

I must confess.....I was poking the bear. I have worked with LN2 for 25+ years and personally believe the risk of asphyxiation is highly over exaggerated.  Any room with adequate ventilation (and that's a very loose term) is safe. Most interactions with an LN2 environment are short term, except perhaps for the worker(s) at the NHSBT National Frozen Blood Bank where special ventilation systems should have been designed into their operation.

I was expecting answers involving cryogenic burns, too.

I'm sure you are right about exaggeration.  This is something that genuinely worries me about Health and Safety (amongst other things).  If they keep making all sorts of claims where things are DANGEROUS, the old thing about "crying wolf" will occur when something that is REALLY DANGEROUS gets ignored, because people get apathetic about H&S because of exaggeration.

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43 minutes ago, Malcolm Needs said:

I'm sure you are right about exaggeration.  This is something that genuinely worries me about Health and Safety (amongst other things).  If they keep making all sorts of claims where things are DANGEROUS, the old thing about "crying wolf" will occur when something that is REALLY DANGEROUS gets ignored, because people get apathetic about H&S because of exaggeration.

Well said. The world is so "dangerous" these days, it's a wonder we're still around.

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On 5/5/2017 at 10:48 AM, exlimey said:

Well said. The world is so "dangerous" these days, it's a wonder we're still around.

The only thing dangerous right now is what's sitting in the WH.

::cough, cough::

...and now, back to our regularly scheduled program.

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On 5/5/2017 at 2:04 AM, Malcolm Needs said:

The SCARF membership used to be held by NHSBT-Tooting Centre, where Alan Gray used to organise units from our own rare donors (by "ours", I mean the NHSBT, not Tooting alone - although most of them were Tooting donors, because of the rare donor screening programme, run by Alan, which found many rare donors).  Since the latest reorganisation of RCI, I do not know who is holding the membership, but I do know that I was told that NHSBT are sending out fewer rare donor samples.  Your own Head of Laboratory may know or, if not, give Alan a ring at Tooting.  SCARF runs on a reciprocal arrangement, so that, if NHSBT does not send in samples itself, it will not receive samples from others.  In addition, and, as far as I know, since the rare donor screening programme has been removed from Tooting RCI and has gone to Filton RCI, the whole thing has ground to a halt, because of pressure of work - but this information may well now be out of date.

I am simply amazed that you are still using liquid nitrogen to freeze and store your rare red cells, for the very reason you give; H&S.

You should have access to a -80oC freezer, as these were being purchased for all Centres not too long ago (certainly, I was still working when this was offered, and I only retired at the end of October 2016), in which case you can go freezing in glycigel.  There is a recipe for this and directions for use in the library section of this site (I know, because I put it on there, although it was written by Alan) under SOPs.  You should be able to get an up-to-date version from QPulse.  Of course, I have made the assumption here that you are working for NHSBT, whereas you may, of course, be working for the Northern Irish, Scottish or Welsh Blood Services, so, if so, if you telephone 0203 123 8300 and ask to speak to Alan Gray (or Doris Lam, as Alan now works part time), you should be able to obtain an SOP.

Hello, 

We have frozen rare cells in glycerol in this lab. Whenever we use these cells, we have to thaw the entire vial, take out some cells and refreeze it (where in LN2 frozen cells never needed to be thawed once frozen). I always wonder what happen to those cells frozen in glycerol once they goes through about 3 cycles of freezing and thawing. I had thrown out a few vials because the cells are all hemolyzed. But I do not think it is because of the age of the cells since I had successfully thawed a few sources that are about 15+ years old. Any thoughts ? 

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These days we don't use glycerol to freeze rare red cells, but glycigel (for the recipe, see the library, user submitted, SOPs), as we find the preservation better and easier.

As far as your findings are concerned, I agree that it is probable nothing to do with the actual age of the red cells.  It is much more likely to be the formation of ice crystals in the red cells over and over again, with successive freezing and thawing episodes.

Essentially, the glycerol acts as an "anti-freeze" (rather like you would find in the water in the radiator of your car).  This glycerol affects the temperature at which water freezes (fairly obviously, from what I have just said!), but also affects the way the water crystals form and how they affect the red cell membrane (they are not as "sharp", for want of another way of putting it).  The use of glycerol does not make the crystal formation absolutely "blunt" however, and so each time an aliquot of red cells is thawed, and then refrozen, there will be more and more damage to the red cell membrane, and this is cummulative.  So, there may be very little haemolysis seen in a sample that was frozen down once, 15 years previously, but only thawed for the first time now, whereas a sample frozen down say, two years previously, but frozen and thawed several times may show gross haemolysis.

All of that having been said, however, the age of the red cells when they are first frozen down can affect recovery (the fresher the cells when frozen, the better the recovery), as can the type of the red cells being frozen down.  For example, Rhnull individuals normally have a well-compensated haemolytic anaemia because of membrane abnormalities leading to stomatocytosis, and these red cells have a "fragile" membrane from the "word go", and so such red cells never recover quite as well as "normal" red cells.

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11 hours ago, Malcolm Needs said:

These days we don't use glycerol to freeze rare red cells, but glycigel (for the recipe, see the library, user submitted, SOPs), as we find the preservation better and easier.

As far as your findings are concerned, I agree that it is probable nothing to do with the actual age of the red cells.  It is much more likely to be the formation of ice crystals in the red cells over and over again, with successive freezing and thawing episodes.

Essentially, the glycerol acts as an "anti-freeze" (rather like you would find in the water in the radiator of your car).  This glycerol affects the temperature at which water freezes (fairly obviously, from what I have just said!), but also affects the way the water crystals form and how they affect the red cell membrane (they are not as "sharp", for want of another way of putting it).  The use of glycerol does not make the crystal formation absolutely "blunt" however, and so each time an aliquot of red cells is thawed, and then refrozen, there will be more and more damage to the red cell membrane, and this is cummulative.  So, there may be very little haemolysis seen in a sample that was frozen down once, 15 years previously, but only thawed for the first time now, whereas a sample frozen down say, two years previously, but frozen and thawed several times may show gross haemolysis.

All of that having been said, however, the age of the red cells when they are first frozen down can affect recovery (the fresher the cells when frozen, the better the recovery), as can the type of the red cells being frozen down.  For example, Rhnull individuals normally have a well-compensated haemolytic anaemia because of membrane abnormalities leading to stomatocytosis, and these red cells have a "fragile" membrane from the "word go", and so such red cells never recover quite as well as "normal" red cells.

Thank you very much for the explaination. It is very helpful. We are very lucky to have a resourceful mentor like yourself who is here to answer any questions that we have. I am checking out the recipe of glycigel in the library and I have a couple of questions. 

1) What ratio of red cells to glycigel is appropiate? Do you warm up the gel before suspending red cells into it? If so, we have to keep the red cell suspended gelatin warm till we freeze the sample? 

2) Can I make red cell droplets in liquid nitrogen using glycigel suspended red cells and keep the vials in -80C freezer? (since we do not have long term nitrogen storage here). 

3) How would I thaw the glycigel suspended frozen red cells? Do I just suspend it in warm 0.9%saline? 

Edited by dothandar

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1 hour ago, dothandar said:

Thank you very much for the explaination. It is very helpful. We are very lucky to have a resourceful mentor like yourself who is here to answer any questions that we have. I am checking out the recipe of glycigel in the library and I have a couple of questions. 

1) What ratio of red cells to glycigel is appropiate? Do you warm up the gel before suspending red cells into it? If so, we have to keep the red cell suspended gelatin warm till we freeze the sample? 

2) Can I make red cell droplets in liquid nitrogen using glycigel suspended red cells and keep the vials in -80C freezer? (since we do not have long term nitrogen storage here). 

3) How would I thaw the glycigel suspended frozen red cells? Do I just suspend it in warm 0.9%saline? 

Opps my appology. I just saw the recipe for "Brentwood" solution for thawing.

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10 hours ago, dothandar said:

Thank you very much for the explaination. It is very helpful. We are very lucky to have a resourceful mentor like yourself who is here to answer any questions that we have. I am checking out the recipe of glycigel in the library and I have a couple of questions. 

1) What ratio of red cells to glycigel is appropiate? Do you warm up the gel before suspending red cells into it? If so, we have to keep the red cell suspended gelatin warm till we freeze the sample? 

2) Can I make red cell droplets in liquid nitrogen using glycigel suspended red cells and keep the vials in -80C freezer? (since we do not have long term nitrogen storage here). 

3) How would I thaw the glycigel suspended frozen red cells? Do I just suspend it in warm 0.9%saline? 

I will touch base with Alan Gray (who is the real expert in this field - you will see, that it was he who wrote the SOPs) and get your questions answered, but, as both he and I are officially retired, it might take a couple of days for us to contact one another.

Thank you for your kind comments.

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16 hours ago, Malcolm Needs said:

I will touch base with Alan Gray (who is the real expert in this field - you will see, that it was he who wrote the SOPs) and get your questions answered, but, as both he and I are officially retired, it might take a couple of days for us to contact one another.

Thank you for your kind comments.

Thank you very much. Cant wait to play with this glycigel 

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On 30/09/2017 at 1:02 AM, dothandar said:

Thank you very much. Cant wait to play with this glycigel 

It would appear that Alan is on annual leave outside the UK at the moment, but don't give up.  He has to come back at some time (unless he has won the national lottery)!

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dothandar, as I thought, Alan has been on holiday, however, he has now returned, and here are his answers to your queries:

"Hi Malcolm,

Sorry for the delay, just returned from a month in Zimbabwe!

Glycigel is solid at 4oC in storage, but thaws readily at room temperature for use.  A 1:1 ratio of red cells to glycigel is normal.  Mix the cells into the glycigel and then immediately freeze the tube at -80oC.

Don't understand 2.  Glycigel is a way of preserving red blood cells to obviate the need for liquid nitrogen, so why freeze glycigel suspended red cells in liquid nitrogen?

If the red cell shave previously been frozen in liquid nitrogen, then they can be transferred to -80oC storage and should recover well.

Recovery of glycigel suspended red cells requires washing in dextrose, and then saline, until haemolysis stops.  I think we used 50%, then 10% and then saline."

I hope that helps.

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On ‎10‎/‎11‎/‎2017 at 2:40 AM, Malcolm Needs said:

dothandar, as I thought, Alan has been on holiday, however, he has now returned, and here are his answers to your queries:

"Hi Malcolm,

Sorry for the delay, just returned from a month in Zimbabwe!

Glycigel is solid at 4oC in storage, but thaws readily at room temperature for use.  A 1:1 ratio of red cells to glycigel is normal.  Mix the cells into the glycigel and then immediately freeze the tube at -80oC.

Don't understand 2.  Glycigel is a way of preserving red blood cells to obviate the need for liquid nitrogen, so why freeze glycigel suspended red cells in liquid nitrogen?

If the red cell shave previously been frozen in liquid nitrogen, then they can be transferred to -80oC storage and should recover well.

Recovery of glycigel suspended red cells requires washing in dextrose, and then saline, until haemolysis stops.  I think we used 50%, then 10% and then saline."

I hope that helps.

Thank you very much! This is very helpful to see that cells that are previously frozen in liquid nitrogen can be transferred to -80C and it would recover well.

I should have been more clear in my question (#2) above. What I envisioned was making droplets in liquid nitrogen and storing the vials with droplets (instead of an aliquot) to avoid having to thaw and refreeze the same vial every time we use the cells. The plan was to take one frozen droplet out of the vial and use it instead of thawing the entire vial to take out a drop. I assume Alan's statement about previously frozen in liquid nitrogen and recovering well in -80C storage implied that we can make droplet in liquid nitrogen using glycigel and store them in -80C long term?

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5 hours ago, dothandar said:

Thank you very much! This is very helpful to see that cells that are previously frozen in liquid nitrogen can be transferred to -80C and it would recover well.

I should have been more clear in my question (#2) above. What I envisioned was making droplets in liquid nitrogen and storing the vials with droplets (instead of an aliquot) to avoid having to thaw and refreeze the same vial every time we use the cells. The plan was to take one frozen droplet out of the vial and use it instead of thawing the entire vial to take out a drop. I assume Alan's statement about previously frozen in liquid nitrogen and recovering well in -80C storage implied that we can make droplet in liquid nitrogen using glycigel and store them in -80C long term?

I assume that too.

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