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Lingkwyz

General Questions/Clarifications in BloodBanking

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Hi Lingkwyz,

Well, in the case of a weak A and a strong B in a newborn, once again, it is impossible to say at the moment.  It could be a genuine weak A phenotype, or, it could be, in this case, the B-transferase "winning" against the A-transferase in direct competition.

I am VERY jealous of your patient with suspected anti-PP1Pk; I haven't seen one of those for years now!

Best wishes,

Malcolm

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I would like to come back to your question about neutral cards not containing AHG. (And apologies for the delay, have been inundated with my day to day work recently).   So - IgG antibodies can not usually agglutinate red cells directly.  They stick on to the red cells and can cause their destruction, but for us to be able to see that the IgG antibodies are present, we need agglutination.  So in order to make IgG antibodies agglutinate, we have to add something that will help them.  This 'something else' is either AHG or enzymes.  The two react in different ways.  The AHG makes a bridge between the IgG molecules attached to the red cells; and the enzymes remove the outer negatively-charged layer of the red cell membrane so that the distance between them is reduced and IgG molecules can agglutinate directly.  So, for the AHG test, you need a source of AHG. In the old days (and still in many places today), where the test was done in tubes, this was added to the test after incubation and washing as a liquid.  In gel, the AHG is already in the cards.  So the 'helper' is present in the gel.  For enzymes, the 'helper' is already in the cells, which have been pre-treated with enzymes.  Therefore you do not need to have a second helper in the gel.  If you put AHG in the gel as well as using enzyme treated cells you are using two different helpers at the same time, when for most cases one is enough.  You can choose to use both in an enzyme Coombs technique; it can be helpful for identifying Kidd antibodies; but it is not a good idea to use this as a routine technique unless you want to increase considerably the number of identification panels you have to do, most of which will lead to nothing.

Hope that helps

anna

 

 

 

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I would say that the two "enhancement" methods mentioned (enzyme and AHG) above are more complimentary than interchangeable.  It also depends on the method. For example, our Papain tube treatment method finishes with an AHG step.

Scott

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9 hours ago, Malcolm Needs said:

Hi Lingkwyz,

Well, in the case of a weak A and a strong B in a newborn, once again, it is impossible to say at the moment.  It could be a genuine weak A phenotype, or, it could be, in this case, the B-transferase "winning" against the A-transferase in direct competition.

I am VERY jealous of your patient with suspected anti-PP1Pk; I haven't seen one of those for years now!

Best wishes,

Malcolm

Weak ABO:

Oh. Then it could be a multitude of other possibilities. So, as routine lab, it might happen  that a newborn be "temporarily mis-typed" in cases of weak or subgroups of ABO. As they come of age, they could probably be corrected. Am I getting it right ?

 

Anti-PP1Pk:

Interesting as it may seem, the sample had been sent to a reference lab. Confirmed as Anti-PP1Pk. Open and shut case as it may seem. But the curious cat was at it again:

6 year old female 

DX: Congestive heart disease.

Blood group: Indeterminate

Solid phase testing:

Anti-A : 4+

Anti-B : 0 

Anti-D: 4+

Rh Ctrl: 0

A1 Cells: 4+

B Cells : 4+

Ab ID: Confirmed Anti-PP1Pk from reference lab

-"Indeterminate" for 6 years. 1st testing 2011 (18 months of age)

^^

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9 hours ago, galvania said:

I would like to come back to your question about neutral cards not containing AHG. (And apologies for the delay, have been inundated with my day to day work recently).   So - IgG antibodies can not usually agglutinate red cells directly.  They stick on to the red cells and can cause their destruction, but for us to be able to see that the IgG antibodies are present, we need agglutination.  So in order to make IgG antibodies agglutinate, we have to add something that will help them.  This 'something else' is either AHG or enzymes.  The two react in different ways.  The AHG makes a bridge between the IgG molecules attached to the red cells; and the enzymes remove the outer negatively-charged layer of the red cell membrane so that the distance between them is reduced and IgG molecules can agglutinate directly.  So, for the AHG test, you need a source of AHG. In the old days (and still in many places today), where the test was done in tubes, this was added to the test after incubation and washing as a liquid.  In gel, the AHG is already in the cards.  So the 'helper' is present in the gel.  For enzymes, the 'helper' is already in the cells, which have been pre-treated with enzymes.  Therefore you do not need to have a second helper in the gel.  If you put AHG in the gel as well as using enzyme treated cells you are using two different helpers at the same time, when for most cases one is enough.  You can choose to use both in an enzyme Coombs technique; it can be helpful for identifying Kidd antibodies; but it is not a good idea to use this as a routine technique unless you want to increase considerably the number of identification panels you have to do, most of which will lead to nothing.

Hope that helps

anna

 

 

 

Hi anna!

Great to hear from you! You seem to be into the gel technique. The way you explained was so clear!

So anna, if adding enzyme to the mix would be sufficient as "helper" for the agglutination, why do we still add Anti-IgG to the tube method testing of enzyme-treated cells? As I experience, the tube method was and still is now doing the same? Only in gel that we get the chance to "omit" the AHG and do the test "Non-IAT". So, I started to presume that there must be something in the "gel" or the technique that grants us to do so.

 

Thanks Anna.

 

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19 hours ago, Lingkwyz said:

Weak ABO:

Oh. Then it could be a multitude of other possibilities. So, as routine lab, it might happen  that a newborn be "temporarily mis-typed" in cases of weak or subgroups of ABO. As they come of age, they could probably be corrected. Am I getting it right ?

 

Anti-PP1Pk:

Interesting as it may seem, the sample had been sent to a reference lab. Confirmed as Anti-PP1Pk. Open and shut case as it may seem. But the curious cat was at it again:

6 year old female 

DX: Congestive heart disease.

Blood group: Indeterminate

Solid phase testing:

Anti-A : 4+

Anti-B : 0 

Anti-D: 4+

Rh Ctrl: 0

A1 Cells: 4+

B Cells : 4+

Ab ID: Confirmed Anti-PP1Pk from reference lab

-"Indeterminate" for 6 years. 1st testing 2011 (18 months of age)

^^

Tried to resolve the reverse grouping discrepancy.. Adsorbed patient's plasma with known P1+ reagent red cell using Method 3-12 of the AABB Technical Manual 17th edition.

Results:

A1 Cell: 0

B Cell: 3+

P1+ cell: 0

 

-The results were not officially reported for we don't have a "local" procedure on Adsorption Techniques. Nevertheless, I had to cure my itch and well, it really paid off. The patient's blood group was resolved in my own self but officially, the patient is still "Blood Group Indetermindate" ^^

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-+Okay - so going back to AHG and enzymes in answer to your last question:  Enzymes will destroy a large number of antigens, so you would not be able to pick up most of the 'normal' antibodies to antigens in the Duffy and MNS blood group systems.  So you can never JUST do an enzyme technique.  On the other hand some antibodies will be enhanced using enzyme techniques; that used to be much more important in the 'old days' when the AHG reagents were less sensitive than they are today.  Now AHG reagents will (should!) pick up very low levels of clinically significant antibodies; for example they are  (should be!) standardised to be able to detect anti-D at 0.05IU/ml, which is extremely low indeed.  As Malcolm has already said in a previous post, most enzyme-only antibodies are not clinically significant.

So, what this means is that you can do an IAT on its own; or you can do an IAT technique and an enzyme technique; or you can do an IAT and an enzyme-IAT.  Regulations change from country to country as to whether an enzyme technique for the antibody screen is a requirement or not.  What you cannot do is JUST an enzyme technique or JUST an enzyme-IAT technique or even a combination of these two.  When you add AHG to the enzyme tube test, you are effectively carrying out an enzyme-IAT.  As long as this is not being done instead of a simple IAT, then that's fine but is definitely not common practice everywhere.  Also, bear in mind that for antibody screens, tube is not as sensitive as gel.....

Then for your indeterminate group.  You have a positive reverse group in a patient with an anti-PP1Pk.  Well, as all cells will be positive for this antigen, and the antibody is presumably active at room temperature, then her plasma is reacting (and will always react as long as her antibody is visible) with the reverse grouping cells.  On a more urgent note, if she needs transfusion then you are in big trouble.  I would strongly advise you to test her siblings, and close relations (and if she's from a village or area where there's a lot of marrying within cousins, then people from her village too) to see if any of them are compatible.

Malcolm - do you know if there's any frozen blood anywhere? 

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10 minutes ago, galvania said:

Malcolm - do you know if there's any frozen blood anywhere? 

Yes, there are, but anyone wanting to use them would have to go through the IBGRL, as far as I know, as they act as the "gate keepers" for units of rare frozen blood.

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33 minutes ago, galvania said:

-+Okay - so going back to AHG and enzymes in answer to your last question:  Enzymes will destroy a large number of antigens, so you would not be able to pick up most of the 'normal' antibodies to antigens in the Duffy and MNS blood group systems.  So you can never JUST do an enzyme technique.  On the other hand some antibodies will be enhanced using enzyme techniques; that used to be much more important in the 'old days' when the AHG reagents were less sensitive than they are today.  Now AHG reagents will (should!) pick up very low levels of clinically significant antibodies; for example they are  (should be!) standardised to be able to detect anti-D at 0.05IU/ml, which is extremely low indeed.  As Malcolm has already said in a previous post, most enzyme-only antibodies are not clinically significant.

So, what this means is that you can do an IAT on its own; or you can do an IAT technique and an enzyme technique; or you can do an IAT and an enzyme-IAT.  Regulations change from country to country as to whether an enzyme technique for the antibody screen is a requirement or not.  What you cannot do is JUST an enzyme technique or JUST an enzyme-IAT technique or even a combination of these two.  When you add AHG to the enzyme tube test, you are effectively carrying out an enzyme-IAT.  As long as this is not being done instead of a simple IAT, then that's fine but is definitely not common practice everywhere.  Also, bear in mind that for antibody screens, tube is not as sensitive as gel.....

Then for your indeterminate group.  You have a positive reverse group in a patient with an anti-PP1Pk.  Well, as all cells will be positive for this antigen, and the antibody is presumably active at room temperature, then her plasma is reacting (and will always react as long as her antibody is visible) with the reverse grouping cells.  On a more urgent note, if she needs transfusion then you are in big trouble.  I would strongly advise you to test her siblings, and close relations (and if she's from a village or area where there's a lot of marrying within cousins, then people from her village too) to see if any of them are compatible.

Malcolm - do you know if there's any frozen blood anywhere? 

Hi Anna!

Gel Card:

In my experience, which hasn't been that long by the way, enzyme techniques are "supplementary" methods to give addtitional conviction to the antibody in question. (Please pardon me, I'm using my own words.) Never has it been done that one used enzyme techniques "catch" solely and got away with it. It would be too naive for a person to do so.

I was waiting for somebody to say "because Gel Card Technology is more sensitive than Tube Method". It could only be the missing piece. Everyone is telling me so, but no one can answer my follow-up. Anna, please enlighten me why Gel Card Method is more sensitive that Tube. (Ergo, Non-IAT enzyme tests are done on this method.)

 

Anti-PP1Pk:

On her first visit, way back 2011, she was 18 months old back then. The lab (I wasn't here back then!) had to call in her mum, her siblings, and some of her folks. Having incompatible for all of the prospect donors, the hospital had to cancel her Cardiac OR. Now, she's 6 years old, still battling for the cardiac problem and still no donor. (And yes, "inbreeding" is popular to these peeps.)

58 minutes ago, Malcolm Needs said:

Yes, there are, but anyone wanting to use them would have to go through the IBGRL, as far as I know, as they act as the "gate keepers" for units of rare frozen blood.

Hi Malcolm!

Can you probably hook us up with the unit if our director chooses to seek help from IBGRL?

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21 minutes ago, Lingkwyz said:

Hi Malcolm!

Can you probably hook us up with the unit if our director chooses to seek help from IBGRL?

No, I am afraid it is not within my power to so do.

Your Director (and it would have to be your Director, as a minimum) would have to go through the IBGRL.  Probably the best person would be Nicole Thornton (nicole.thornton@nhsbt.nhs.uk), but, be aware that the IBGRL is not open at weekends and English Bank Holidays (public holidays), and it may also take some time to organise getting the donation/donations to your country (and it is not necessarily cheap)!

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20 hours ago, Lingkwyz said:

I was waiting for somebody to say "because Gel Card Technology is more sensitive than Tube Method". It could only be the missing piece. Everyone is telling me so, but no one can answer my follow-up. Anna, please enlighten me why Gel Card Method is more sensitive that Tube. (Ergo, Non-IAT enzyme tests are done on this method.)

Hi

Well let me deal with the 'ergo' part first.  Depending ion the circumstances, enzyme-IATs are ALSO done on gel.  If you have the slightest doubt about Kidd antibodies, this is one of the best methods for finding them.  It is just not very common (but not unknown) for this to be used as a routine method for ALL antibody screens

So why is gel more sensitive than tube.  First of all this ONLY applies to atypical antibodies.  the reason lies in the method.  Firstly, the tube technique tends to be (in most people's hands) very imprecise, with most people using drops (1 or 2 or 4.....) of plasma and drops (1 or 2...) of cells (3 or 4 or 5%.....) with or without LISS; if with LISS then either with LISS addition or by suspension of the cells in LISS; incubating for x minutes where x can be from 5 to 60 mins.  Then the tubes are washed 3 or 4 times with a spin cycle that is xxx(?) rpm for yy minutes - you can fill in your own values, removing some/most/all of the liquid between washes.-  Then to your possibly quite diluted remaining red cells, from which you may have washed off weakly attached antibodies anyway,  you add 1 or 2 drops of AHG and spin with a spin cycle that is xxx(?) rpm for yy minutes then read by eye/with a magnifying glass/with a microscope after re-suspending the cell button from so gently that you can't see anything to so hard that all your weak agglutinates have been shaken away................I will admit the variation in any one site is much less than this, but globally there is just no standardisation.  Gel is standardised, can be done on an instrument, and there is no washing.

But there are still good reasons for gel users to revert to tube techniques from time to time. 

Does that answer your question?

(And yes, I've done literally millions of tube IATs in my time; and no, I would not go back to using them as a routine method EVER!)

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10 hours ago, galvania said:

Hi

Well let me deal with the 'ergo' part first.  Depending ion the circumstances, enzyme-IATs are ALSO done on gel.  If you have the slightest doubt about Kidd antibodies, this is one of the best methods for finding them.  It is just not very common (but not unknown) for this to be used as a routine method for ALL antibody screens

So why is gel more sensitive than tube.  First of all this ONLY applies to atypical antibodies.  the reason lies in the method.  Firstly, the tube technique tends to be (in most people's hands) very imprecise, with most people using drops (1 or 2 or 4.....) of plasma and drops (1 or 2...) of cells (3 or 4 or 5%.....) with or without LISS; if with LISS then either with LISS addition or by suspension of the cells in LISS; incubating for x minutes where x can be from 5 to 60 mins.  Then the tubes are washed 3 or 4 times with a spin cycle that is xxx(?) rpm for yy minutes - you can fill in your own values, removing some/most/all of the liquid between washes.-  Then to your possibly quite diluted remaining red cells, from which you may have washed off weakly attached antibodies anyway,  you add 1 or 2 drops of AHG and spin with a spin cycle that is xxx(?) rpm for yy minutes then read by eye/with a magnifying glass/with a microscope after re-suspending the cell button from so gently that you can't see anything to so hard that all your weak agglutinates have been shaken away................I will admit the variation in any one site is much less than this, but globally there is just no standardisation.  Gel is standardised, can be done on an instrument, and there is no washing.

But there are still good reasons for gel users to revert to tube techniques from time to time. 

Does that answer your question?

(And yes, I've done literally millions of tube IATs in my time; and no, I would not go back to using them as a routine method EVER!)

So basically, because you can control/standardize  the activities within the technique, it makes the technique more sensitive. Let me rephrase. What I'm getting at your input, and please correct me if I'm wrong, is that: minimizing human intervention "intra-procedure" leads to reduced "human error". That makes it more sensitive than tube method? 

Sorry Anna if I'm really stubborn at this but I've got another question: 

The purpose of washing cells in tube method is to eliminate free unbound IgGs which could "neutralize" the AHG, why isn't washing necessary of AHG Cards? (Poly and mono). 

 

Thanks so much Anna.

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On 28 July 2016 at 7:38 PM, Malcolm Needs said:

No, I am afraid it is not within my power to so do.

Your Director (and it would have to be your Director, as a minimum) would have to go through the IBGRL.  Probably the best person would be Nicole Thornton (nicole.thornton@nhsbt.nhs.uk), but, be aware that the IBGRL is not open at weekends and English Bank Holidays (public holidays), and it may also take some time to organise getting the donation/donations to your country (and it is not necessarily cheap)!

Thanks for the details Malcolm.

There's a reason why I don't call the shots and I'm keeping it that way. I'm endorsing details to our director. Whatever he decides on, its all in his hands. Anyway, I got the chance to solve the 6-year old ABO discrepancy. That was already enough for me. (Although they didn't accept it!):P

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On ‎30‎/‎07‎/‎2016 at 1:59 AM, Lingkwyz said:a

The purpose of washing cells in tube method is to eliminate free unbound IgGs which could "neutralize" the AHG, why isn't washing necessary of AHG Cards? (Poly and mono). 

When you do a tube technique, the cells and the plasma are all sitting in the mix together.  If you add AHG to this the plasma is omnipresent, and has probably, after incubation, ended up sitting on top of the red cells, which have sedimented down because heavier - so the plasma is the first thing the AHG comes into contact with.  In gel, the  AHG is in the gel.  You pipette the red cells first, then the plasma, so when you centrifuge, the AHG first comes into contact with the red cells.  So it can react with sensitised cells before coming into contact with the plasma

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1 hour ago, galvania said:

When you do a tube technique, the cells and the plasma are all sitting in the mix together.  If you add AHG to this the plasma is omnipresent, and has probably, after incubation, ended up sitting on top of the red cells, which have sedimented down because heavier - so the plasma is the first thing the AHG comes into contact with.  In gel, the  AHG is in the gel.  You pipette the red cells first, then the plasma, so when you centrifuge, the AHG first comes into contact with the red cells.  So it can react with sensitised cells before coming into contact with the plasma

That was what I had in mind! But of course, I had to confirm it from you. Thanks anna.!

Another question for all..

For Antibody Identification, is an Anti -IgG-only DAT enough to replace autocontrol to differentiate the antibody as allo or auto?

 

Thanks!

 

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1 hour ago, Malcolm Needs said:

NO!

Omaygad! They're implementing it here! Ab ID with DAT (solid) is autocontrol for them! We're dead! 

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I would strongly suggest that they read:  Sachs UJH, Röder L, Santoso S, Bein G.  Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia?  A prospective study of 504 cases.  British Journal of Haematology 2006; 132: 651-661, and tell them not to be fooled by the title!

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Basically Lingkwyz, an auto control looks for antibodies that are present in the patient's plasma that are going to stick on to the patient's red in vitro in the same conditions that you have used to perform your antibody identification.  The DAT looks for antibodies that are present in the patient's plasma that have stuck on to red cells that are present in the patient's circulation in vivo.  Although you often DO get the same results for both, it is not necessarily the case and the results do not have the same significance

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23 hours ago, Lingkwyz said:

Any other references to support my stand?

I would recommend Judd's Methods in Immunohematology (3rd ed). This is actually a 'cookbook' that spells out how to perform many many different tests in blood bank. These tests meet current accepted practice. It is available from AABB - you can order it on line, even as a non-member.

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