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Posts posted by Lingkwyz
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Well, after three pages, I only have eric1980 on our side..
I don't know, but as per my short experience, It kinda suffices the need. Although, if ever you are unlucky, if you happen to come across an antibody to a more frequent antigen, let's say cellano, then problem it is.
PS. - We too keep panels 3 months expired. For exclusion purposes.
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Any other references to support my stand?
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Believe me. It's hard to talk to rocks guys. I've tried. Please give me references.
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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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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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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!)
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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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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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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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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:
On 07/07/2016 at 10:15 AM, Malcolm Needs said:Yes, I can see from where you are coming. It can be so difficult on occasions (which is why I have always maintained that we should be paid our worth)!!!!!!!!!!!!!
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.
- jmphil4, Malcolm Needs and AMcCord
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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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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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20 hours ago, jmphil4 said:
My game plan with the kidds is to honor what antibody I think I see (assuming it agrees with their phenotype). Because titers with kidds are notorious for falling quickly, and their transfusion reactions can be particularly unpleasant, I always think its better to be safe than sorry.
19 hours ago, AMcCord said:Better safe than sorry is my game plan, too.
What I'm getting at your suggestions is that; if the patient has this "Non-specific Reactions", I must assure myself that neither of the Kidd antigens are negative, which means, all "Nonspecific Reactions" must be antigen typed for Kidd.
Am I getting it right guys?
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8 hours ago, Malcolm Needs said:
I'm afraid that the answer to your question is both "yes" and "no" - which is not much help to you, I realise!
Most newborn babies give weaker reactions with ABO antibodies than do slightly older children, due to the fact that the transferase enzymes that "confer" the immunodominant sugar residue onto the backbone molecule do not "work" at their peak performance in a newborn. In very many cases, therefore, a baby who will eventually be a group A1, will group as group A2 at birth. Similarly, an A2 individual can group as a very weak group A individual indeed (and so on and so forth).
All that having been said, you will occasionally come across what have been described as precocious babies. These will not only have adult strength ABO antigens, but can also produce their own IgM ABO antibodies at birth (and we know it is thier own IgM antibody, because, in some cases, the ABO specificity is one that cannot be produced by the mother, and, in others, the Gm type or the Km type does not match that of the mother), and so, in these cases, you will know the adult ABO type from the word go.
This is why I say that the answer to your question is bot "yes" and "no".
Hi Malcolm..
Seems a dilemma. Was really wrong at testing the cord sample with the anti-A1 lectin. Did me no good. Curious as I was, needed to challenge the clause in the reagent insert that said:
"Newborn testing results will not be as expected"
Thanks for injecting the idea of the existence of "precocious babies".
The baby was group AB by the way with 1+ in Anti-A and 4+ in Anti-B on newborn gel card testing. Was being told, again, that these are usual results for AB newborns. (Weaker A antigen expression against B antigen on AB newborns)
Any inputs?
PS- by the way, I got a patient today with a suspected Anti-PP1Pk.
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17 hours ago, jnadeau said:
I believe the Kidds are known to react best using PeG. You didn't indicate what enhancement you used in tube.
The enhancement possible for us was LISS. That's it.
So you mean PEG would be the best to use dealing with Kidd antibodies?
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Another question:
Can you detect, on a routine bloodbank, group A2 on a cord blood or newborn sample?
Reactions:
Sample: Cord Blood
- 1+ reaction with Anti-A (routine)
- 0 reaction with Anti-A1 Lectin (Dolichus biflorus)
Thanks
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Hello
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On 7/20/2016 at 4:19 PM, Lingkwyz said:
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?
Everyone in silent mode? ^^
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2 hours ago, SMILLER said:
You probably know this already, but literature and texts tend to see techniques such as a gel or solid-phase a bit too sensitive. To the point where you may pick something up that is just not going to be clinically significant. You did an enzyme method that was negative--it seems like if anti-Jkb was present in any significant amount you should have seen a positive reaction there.
Having said that, at our lab in a situation like this, we would probably call our reference lab for advice, as they would be the ones we would have do further testing anyway.
Scott
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.
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Anyone?
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35 minutes ago, Malcolm Needs said:
I think that all you need do to make the enzyme-treated cells in cassette for IAT "procedural" is to validate the procedure. As far as I can remember, that's all we did for the entire UK National Health Service Blood and Transplant (NHSBT), and, as we had validated the procedure, our accreditation people, who are known to be as fierce as Bengal tigers, were quite happy.
By the way, I didn't spell "sialic" correctly; as you read more of my posts, you will realise (as have most people who have read any), that spelling is not my greatest forte, (and I spell in UK English, rather than US English)!!!!!!!!!!!!!!!!!
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.
35 minutes ago, David Saikin said:Because that is what they are, neutral - I call them buffered gel. There are no additives, only gel. If you want antiglobulin reactions you have to use anti-IgG or polyahg cards. Those neutral gel cards are just like the portion of your ABD Reverse card that does the back type . . . plasma and cells. You can use them for room temp studies or add your own antisera to cells for ag typing.
Yes sir. My question was: what made these neutral cards so special that it is acceptable to omit the AHG?
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1 hour ago, Malcolm Needs said:
Exactly so - they are "non-IAT" procedures, BUT, the treatment of the red cells with papain, in itself, exposes more antigen epitopes to sensitisation by antibody (as long as the antigen is not papain-sensitive, e.g. the Fy(a) antigen) and, at the same time, strips the red cell of much of the cloud of syalic acid molecules, allowing the red cells to come into closer proximity to one another, and thus allowing agglutination. It is, therefore, another way of potentiating antibody/antigen reactions.
As I say, however, using a neutral card for this method was found, in our hands, to produce less "clean" reactions, which were more difficult to read than using papain-treated red cells in a cassette that did actually contain AHG, whether this be a broad spectrum AHG or a monospecific anti-IgG reagent.
I would want to try using the enzyme-treated cells to AHG-incorporated gel cards but it would be non-procedural in our case and would be out of the manufacturer's instructions. Ergo, mistakes would be under my head.
What got me to this question is that on tube method, we do IAT with enzyme-treated cells but on gel card we don't. Why?
And, I dont know if any solid phase technology brands uses enzyme-treated cells. (Because Immucor says they don't.) ^^
Thanks Malcolm Needs.
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1 hour ago, Malcolm Needs said:
As far as I know, the manufacturer just does not add AHG to their neutral cards in the first place. Are you using BioRad? If so Anna/galvania will know far more about this than do I.
Are you performing your routine IAT in tubes, column agglutination technology (CAT) or solid phase? If it is CAT, then you can just use enzyme-treated red cells into one column (to perform the enzyme-IAT) and non-treated red cells into another (to perform the standard IAT). This way, you will no longer have to order your neutral cards, and so you will no longer have any waste!
What we have here sir is one, Gel Card Technology, two, Solid Phase technology (Neo and Echo) and tube techniques.
I'm just puzzled why Neutral Gel Cards don't have Anti-IgG in the mix. 'Coz by then, Enzyme treated cells done on neutral cards are "Non-IAT" procedures. Please correct me if I'm wrong.
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14 hours ago, Malcolm Needs said:
As far as screening is concerned, you are correct in saying that most countries now only mandate for the screening cells and the patient's plasma to be tested by IAT. VERY However, in many countries, including the UK, there is a recommendation (although not a mandate) to use enzyme-treated red cells, as well as an IAT for antibody identification. I must say that I have found this quite useful in many cases, where I have been able to identify an antibody reacting with papain-treated red cells (we tend to use papain, rather than ficin in the UK) in addition to those reacting by IAT. In all probability, "papain-only" antibodies are not clinically significant (although there are a VERY few exceptions to this in the literature), but it is still worthwhile to try to avoid the cognate antigen, so that the antibody is not boosted, and so becomes clinically significant.
All that having been said, we now use papain-treated red cells in an IAT (as well as, obviously, a "normal" IAT with untreated red cells), as we find we get far "crisper" reactions, and fewer non-specific "rubbish" reactions!
Using enzyme-treated cells to do screening is not mandated in our institution as well, but having the reagents expire without use makes me cringe. I may be one of the few doing this procedure and I find it really helpful.
My trouble is, as I noticed, doing enzyme-treated cells on tube involves the addition of the antihuman globulin while the gel card method only involves the neutral buffered gel.
Is there any special characteristic of the neutral buffered gel to eliminate the AHG off the mix?
Thanks
Sudden Onset Hemolysis with weakened D antigen
in Immunohematology Reference Laboratories
Posted
Could it be because of warm autoantibodies "saturating" the D epitopes? We see a lot of WAIHA cases from where I work and the suppression of antigens , especially D, which D-like specificites are far too common. After subjecting these patients with immunosuppressants, their antigen test remarkably improve as well.