Lingkwyz

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

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!)

Is your patient male or female - not that it matters that much,  to be honest???!!!!!!!
IgM antibodies, which include ABO and H isoagglutinins, react optimally at 4oC, and so, if you are storing the patient's sample for 15 minutes in a refrigerator, you will be bringing down the temperature of the plasma in the sample, perhaps not to 4oC, but certainly closer to 4oC.  In addition, IgG ABO and H isoagglutinins also react at 4oC, with no need for any potentiators, such as enzyme-treatment of the red cells, and cause direct agglutination (just see an EDTA sample of a cord blood from a baby suffering from even mild HDFN due to ABO, that has been left in a fridge over night.  The baby cannot have made an IgM auto-antibody at this stage, the maternal IgM antibodies cannot have passed through the placenta, and so the agglutination seen in the sample must be down to the maternal IgG ABO and H isoantibodies).
 
So, to cut a long story short, The patient's condition can account for the reverse group at initial testing, but the temperature can certainly account for the "appearance" of the reaction after time in the fridge.

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)!

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!)

I will try to collate all your inputs in one word: "PRAGMATISM".
I would rather say that as I learn from you guys, I always bump back into this word. Well.. wait! Hold your horses.. I might get a double-dosed sermon with this, but let me try to explain further:
Although there are SOPs, AABB Methods, Guidelines, PPs, (Policies and Procedures), I could only imagine how you do your stuff in the field we all love. Its that: "Hold my beer, I got this!" moment that really starts it all, then comes the protocol for Kidd, or the necessity of  enhancement techniques or the pesky procedures for adsorptions.
If I may quote Malcolm's reply on one of my started  threads:
 
The "worth" this great person might be pertaining to is that: Wait-a-minute.. I-know-this-antibody-moment, or the "Oh I see you Mister antibody.." moment plus the eureka dance..
Though its  the science is what brings us together, I just love the way you how share your approach and "hunches" to our field. I might be too premature to this field myself. I need to develop that 3rd eye. That  stare to the antigram that looks beyond to the pluses and minuses. Nevertheless, I learned a lot from all of you guys.

Give it about 10-20 years playing with the things and listening to people like Malcolm - you'll get there too!  

-+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? 

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.

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" ^^

I will try to collate all your inputs in one word: "PRAGMATISM".
I would rather say that as I learn from you guys, I always bump back into this word. Well.. wait! Hold your horses.. I might get a double-dosed sermon with this, but let me try to explain further:
Although there are SOPs, AABB Methods, Guidelines, PPs, (Policies and Procedures), I could only imagine how you do your stuff in the field we all love. Its that: "Hold my beer, I got this!" moment that really starts it all, then comes the protocol for Kidd, or the necessity of  enhancement techniques or the pesky procedures for adsorptions.
If I may quote Malcolm's reply on one of my started  threads:
 
The "worth" this great person might be pertaining to is that: Wait-a-minute.. I-know-this-antibody-moment, or the "Oh I see you Mister antibody.." moment plus the eureka dance..
Though its  the science is what brings us together, I just love the way you how share your approach and "hunches" to our field. I might be too premature to this field myself. I need to develop that 3rd eye. That  stare to the antigram that looks beyond to the pluses and minuses. Nevertheless, I learned a lot from all of you guys.

I will try to collate all your inputs in one word: "PRAGMATISM".
I would rather say that as I learn from you guys, I always bump back into this word. Well.. wait! Hold your horses.. I might get a double-dosed sermon with this, but let me try to explain further:
Although there are SOPs, AABB Methods, Guidelines, PPs, (Policies and Procedures), I could only imagine how you do your stuff in the field we all love. Its that: "Hold my beer, I got this!" moment that really starts it all, then comes the protocol for Kidd, or the necessity of  enhancement techniques or the pesky procedures for adsorptions.
If I may quote Malcolm's reply on one of my started  threads:
 
The "worth" this great person might be pertaining to is that: Wait-a-minute.. I-know-this-antibody-moment, or the "Oh I see you Mister antibody.." moment plus the eureka dance..
Though its  the science is what brings us together, I just love the way you how share your approach and "hunches" to our field. I might be too premature to this field myself. I need to develop that 3rd eye. That  stare to the antigram that looks beyond to the pluses and minuses. Nevertheless, I learned a lot from all of you guys.

Not all 'nonspecific reactions' must be antigen typed for Kidd antigens. Evaluate the reactions you see - what do they have in common? Are the reactions occurring where a Kidd antigen is present, especially where it is expressed as a double dose (example: Jka+Jkb-) and not occurring where Kidd antigens are not present. Are the single dose cells (example: Jka+Jkb+) not reacting - which could be the case with an antibody that has a low titer. Keep in mind that some antigen positive cells may not react with an antibody with a low titer, even the double dose cells may not react. If this is what you see, and it looks anti-Jka-ish or anti-Jkb-ish, I would antigen type the patient for the appropriate Kidd antigen.
Evaluate your reactions in this way for any antibody that can show dosage, not just potential Jk antibodies, though they are notorious for behaving this way and notorious for causing delayed reactions. You might also see this sort of behavior with newly forming antibodies whose titers are just starting to rise.

I agree with the above.  Non-specific reactions of any kind cannot be ignored out of hand.  You must be careful as with everything in the Lab.  Any set of non-specific reactions could indicate an emerging antibody as someone mentioned above.  That is why we end up doing an AHG crossmatch when we have such patients, even though there are no other specific allo-antibodies present.
Scott
 

I could not agree more.
You will see in many text books that there are 14, 000 Kidd antigens per red cell, BUT, if you read around, this is a mean number, with some workers finding as many as 32, 000 antigen sites per red cell, while others as few as 7, 000 antigen sites per red cell.  Now, all of these are "estimates" and, of course, it also depends upon the expression of the Kidd antigens (or any other antigens, come to that) of the individual being studied.
Let us think for the minute, therefore, about the red cells represented on any antibody identification panel.  They will be, to all intents and purposes, representative of the general population (save for the fact that they are often selected because they express homozygosity for many antigens, and because they complement the other red cells represented in the panel, so that the panel is "useful") and so some of the red cells will express the Kidd antigens at the higher end of the scale, and some at the lower end of the scale.  Those at the lower end of the scale are those about which AMcCord is talking (I think!).

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

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

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
 
 
 

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)
^^

I see this as a current dilemma in our lab since our "seniors" are too fond of the "Nonspecific Reaction" dump. And, if you have read my previous post tilted: "Missing Plasma Protocol", we have faced a patient who had a transfusion reaction which apparently had Anti-Jka but was misdiagnosed as a Nonspecific reaction. I would want to ask for ideas so I could ,at least, evade this problem.
 
Thanks Smiller.

Don't know what you mean Mabel:sarcasm:!!!!!!!!!!!!!!
I would go with testing the father against the mother's plasma/serum first. Even if the mother and father are ABO incompatible, you could always use blood group substance to take out the mother's ABO antibodies or, better still, as this may dilute out such a weak antibody, adsorb the mother's antibody onto the screening cell, and then elute it. This will leave the putative low-incidence antibody free of maternal ABO antibodies.
If then the father's red cells react against the isolated antibody, then I would pull out all the plugs to identify the specificity. As you say, this antibody could have been formed in the previous pregnancy (or in this pregnancy) and could get stronger (and more clinically significant) during the pregnancy.
Identification of any such antibody is a pain, but identification may help predict if it has the potential to cause haemolytic disease It could be one that is known to adsorb onto the apical surface of the placenta, such as an antibody in the Cromer Blood Group System, in which case there should be no more worries, but it could equally be one that has been known to cause problems in the past.
If dad doesn't react with the isolated antibody, then, hey, life's a dream and I wouldn't take it any further.
I'm always more worried about potential HDNF in these cases than transfusion.
Hope this helps (even if I'm not pregnant myself)!!!!!!!!!!!!!!!!!
:D:D:D:D

I saw the "syalic" acid typo, but ain't got no grammar nazi in my blood.. ^^
Living in the lower end of the food chain in our lab, as I am right now, I can't call the shots. And suggesting far off procedures are quite taboo in these kind of people. (As you may now realize, I'm not a Saudi.) I would loveto see one day that these practices of yours becomes a procedure here, or eventually I get to work with an institution which is following one. 
Thanks.
 
 
Yes sir. My question was: what made these neutral cards so special that it is acceptable to omit the AHG?

Just ran it for curiousity.. Anyway, gel was our mainstream method before we had solid phase. We got the solid phase around a year ago.
 
Thanks for the clarification Malcolm.. Will do read on that reference.. Now I can rest in peace.. ^^

I could not agree more Malcolm.. True that!!!
 
As I see, and I hope everyone agrees, working on a "routine" blood bank must have its own system "traps". Cruising through a day of "regular" patient samples sometimes gets you to overlook really tricky ones. In our institution, I would say that we are benefiting on the "Non-specific Reaction" dump. Anything unequivocally an antibody would fall to that dump. Sadly, the situation above fell into that.
 
As per the fee, nah! I always tell my self that I will be here for the science. Sadly again, my wallet don't feel the same!
 

Hi Malcolm Needs!
 
I do agree that prophylactically we must give antigen negative units to suspected antibodies, proven or not. But to this situation, the results gave negative reactions to two Jka positive cells in Solid Phase panel and no reaction to Gel Card both regular and enzyme. Also, this patient have had multiple testings in our lab, all of which were interpreted as  "Non-specific" reactions.
I also had to point out that as a person seeing the result, what would be my indicator for the validity of the Pre Transfusion Gel Card results (which were all negative) which was also taken into consideration, thus the "Non-specific" reaction interpretation.
Thanks for the enlightenments guys and hope to learn more from you guys..