Hi guys.. It's me again.

 

Would like to start this topic for the multitude of questions popping through my mind as start my life in Bloodbanking. I know a lot of you guys  (ehem! you know who you guys are..) are pretty much on the high level bloodbanking, whether skills or knowledge. So, can you spare this chap some of them knowldedge?? ^^

 

Anyway, lemme start;

I recently had one patient 21 yo female, O Neg, Absc Positive with solid phase. Ab ID in solid phase gave a reaction pointing to Anti-D with reactions as low as 1+, max 2+. 

Confirmed the testing through gel card which gave me regular results: weak pos, enzyme: 4+ "mixed-field".

As I was the neophyte, I have been told that this is a "classic" picture of a "Passive Anti-D".

 

So my question is: Why the mixed field? Why didn't the "passive Anti-D" react to all the cells of the D+ reagent red cells?

 

Answers are highly appreciated.

 

 

Thanks

58 minutes ago, Lingkwyz said:

As I was the neophyte, I have been told that this is a "classic" picture of a "Passive Anti-D".

There is NO such thing as a "classic" picture of a "Passive Anti-D".  Whomsoever told you this is a danger.

I know that our (UK) Guidelines do not apply in Saudi Arabia, but I would urge you to have a look at the BCSH Guidelines on antibody testing in pregnancy and the RCOG Green Top Guideline Number 65, on the same subject, by putting these into your search engine, and they both will confirm what I say (I KNOW the BCSH one will, because I was one of the authors!!!!).

 

1 hour ago, Malcolm Needs said:

There is NO such thing as a "classic" picture of a "Passive Anti-D".  Whomsoever told you this is a danger.

I know that our (UK) Guidelines do not apply in Saudi Arabia, but I would urge you to have a look at the BCSH Guidelines on antibody testing in pregnancy and the RCOG Green Top Guideline Number 65, on the same subject, by putting these into your search engine, and they both will confirm what I say (I KNOW the BCSH one will, because I was one of the authors!!!!).

 

But how do you REALLY feel Malcolm?!  LOL......just giving you a hard time.

Brenda Hutson

There, and I thought I had disguised my feelings so well Brenda!!!!!!!!!!!!!!!!!!!!!!!!!!

 

2 hours ago, Lingkwyz said:

Hi guys.. It's me again.

 

Would like to start this topic for the multitude of questions popping through my mind as start my life in Bloodbanking. I know a lot of you guys  (ehem! you know who you guys are..) are pretty much on the high level bloodbanking, whether skills or knowledge. So, can you spare this chap some of them knowldedge?? ^^

 

Anyway, lemme start;

I recently had one patient 21 yo female, O Neg, Absc Positive with solid phase. Ab ID in solid phase gave a reaction pointing to Anti-D with reactions as low as 1+, max 2+. 

Confirmed the testing through gel card which gave me regular results: weak pos, enzyme: 4+ "mixed-field".

As I was the neophyte, I have been told that this is a "classic" picture of a "Passive Anti-D".

 

So my question is: Why the mixed field? Why didn't the "passive Anti-D" react to all the cells of the D+ reagent red cells?

 

Answers are highly appreciated.

 

 

Thanks

Me just being curious... but was wondering why all the additional testing after initial solid phase results?  

14 hours ago, R1R2 said:

Me just being curious... but was wondering why all the additional testing after initial solid phase results?  

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.

 

15 hours ago, Malcolm Needs said:

There is NO such thing as a "classic" picture of a "Passive Anti-D".  Whomsoever told you this is a danger.

I know that our (UK) Guidelines do not apply in Saudi Arabia, but I would urge you to have a look at the BCSH Guidelines on antibody testing in pregnancy and the RCOG Green Top Guideline Number 65, on the same subject, by putting these into your search engine, and they both will confirm what I say (I KNOW the BCSH one will, because I was one of the authors!!!!).

 

Thanks for the clarification Malcolm.. Will do read on that reference.. Now I can rest in peace.. ^^

were all your gel results mf?  sometimes poor cell addition can lead to that in the tubules when the cells fall down out of the reaction area.

Was the mf only in the enzymed cells?  could indicate a cold ab also.

58 minutes ago, David Saikin said:

were all your gel results mf?  sometimes poor cell addition can lead to that in the tubules when the cells fall down out of the reaction area.

Was the mf only in the enzymed cells?  could indicate a cold ab also.

Well, that would be a long shot sir since all roads point out to Passive anti-D (The history of RhIg, childbearing age female, Rh Neg etc.). Also, the cells reacted to D+ reagent red cells only.

Moving on:

What would be the expanation why Neutral Cards used in Ab ID for enzyme-treated cells does not contain any antihuman Globulin?

-As I understand, antibody screening and identification revolves around IAT.. 

Pls enlighten.. ^____^

Sometimes strong, direct agglutinins can cause that mix field appearance in gel like anti M, colds, and rouleaux.    Also, since RHIG is not a "real antibody" it may not react the same as a true antibody.   Ficin in gel can give weird reactions too, IMO.  

Edited July 19, 20169 yr by R1R2

28 minutes ago, R1R2 said:

Sometimes strong, direct agglutinins can cause that mix field appearance in gel like anti M, colds, and rouleaux.    Also, since RHIG is not a "real antibody" it may not react the same as a true antibody.   Ficin in gel can give weird reactions too, IMO.  

The reagent cells were papainized sir. What weird reactions did you experience with ficin-treated cells sir?

I feel that if you don't have a lot of experience with ficin then it can lead you down wrong paths and you waste a lot of time.   Also, it tends to pick up things you don't want to see like colds, Lewis.   Don't get me wrong, it definitely has its place in the BB, just not routinely.   I assumed you were using ficin,  I just read that you are using papain.  

21 minutes ago, R1R2 said:

I feel that if you don't have a lot of experience with ficin then it can lead you down wrong paths and you waste a lot of time.   Also, it tends to pick up things you don't want to see like colds, Lewis.   Don't get me wrong, it definitely has its place in the BB, just not routinely.   I assumed you were using ficin,  I just read that you are using papain.  

The other thing it can depend on is who does the papain or ficin treatment.  It is easy to over-treat the red cells (far easier than to under-treat them, under normal circumstances), which can lead to false positive agglutination.  This is one of the many reasons why the Low's layering one-stage technique is now almost totally redundant.  However, I did use it when I started out on my journey in this wonderful profession, and you could tell that the red cells were over-treated by the fact that they became purple cells!!!!!!!!!!!!!

2 hours ago, Lingkwyz said:

Moving on:

What would be the expanation why Neutral Cards used in Ab ID for enzyme-treated cells does not contain any antihuman Globulin?

-As I understand, antibody screening and identification revolves around IAT.. 

Pls enlighten.. ^____^

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!

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

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!

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.

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.

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.

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)!!!!!!!!!!!!!!!!!

23 hours ago, Lingkwyz said:

Moving on:

What would be the expanation why Neutral Cards used in Ab ID for enzyme-treated cells does not contain any antihuman Globulin?

-As I understand, antibody screening and identification revolves around IAT.. 

Pls enlighten.. ^____^

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. 

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?

Edited July 20, 20169 yr by Lingkwyz

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? ^^

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

Edited July 25, 20169 yr by Lingkwyz
typo

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".

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.