Antibody ID

[Johnny]

Can someone tell me what the requirements are for performing antibody identification. We want to do a ten cell panel and finalize it with the Fishers Exact method to confirm antibody using a 3 positive 3 negative cells. Wondering if this enough. Thanks:)

[Malcolm Needs ☆]

Hi Johnny,

I replied to you, but it has come out on "Screening Antigen Negative Units???" for some reason!

Malcolm:confused:

[BloodBank]

Per Technical Manual, 16th Edition, "When licensed reagents are not available, expired reagents or stored serum specimensfrom patients or donors may be used, provided that control tested on the day of use are acceptable".

When I performed CAP inspection for one facility, supervisor had outdated anti-Jka in stock, and per her she considered it as rare because now a days the cost of each 3 or 5 mL vials is almost 300 or 400 dollars. So she was using outdated anti-Jka as long as control works.

Is this acceptable practice? What can you consider acceptable or not acceptable. We used to keep outdated anti-Kpa, anti-k, anti-U from patient etc.

Any comments?

[Malcolm Needs ☆]

I cannot see any problem with this for screening purposes, but I would have huge problems with the idea if the outdated antisera were used to actually type patients or any units of blood destined to be transfused to a patient with anti-Kpa, anti-k, anti-U, etc, unless they were then fully checked with a licensed reagent. I am fully aware that these reagents can be/are expensive, but I also think that, if they are available, and something went wrong, and they had not been used, there could be big trouble!

[Dr. Pepper]

Hi Johnny - Keep in mind that you are never totally confirming an ID with the 3+/3- cells, but rather reaching a 95% confidence level that your interpretation is correct (I always wondered who died and left Mr. Fisher in charge with his 1 in 20 standard...). This may not always be possible due to inherent shortcomings in panel antigen composition; the current AABB tech manual (p. 473) discusses alternative probability models. A very good rule of thumb, born out by the probability math, is to never to base your ID on the reactions with a single cell - find a few more e-, Cw+ etc. cells to confirm the ID. This will also allow elimination of more antibody possibilities when dealing with antibodies to high-incidence antigens. We buy 3 different panel lots a month, both for test volume and antigenic diversity.

[Cathy]

Johnny, Has your question been answered? Are you asking for help in ruling out antibodies or other aspects of testing?

[Johnny]

Johnny, Has your question been answered? Are you asking for help in ruling out antibodies or other aspects of testing?

Sort of. What a want to know is weather or not the "rule out" method is good enough when it comes to antibody identification or do we need to perform other test as a requirement. The "rule out" method appears to be pretty conclusive most of the time, specially when I am dealing with a single antibody. I am wondering, if by continuing testing by performing the Fisher exact method, I have a better probability and not miss other antibodies. Thanks for your help.

[David Saikin]

to malcolm

The FDA and CAP allow the use of outdated "rare" antisera for antigen typing, the only caveat is that you have to run positive and negative controls. The only antisera that MUST be used indated are the ABO and Rh(o)D.

[David Saikin]

Johnny

With only one 10 cell panel you will probably have a difficult time finding 3 neg and 3 pos cells for certain clinically significant antibodies or if you have multiples. It works great with one panel if you only have an anti-E or an anti-K. It can get dicey if there is a Fy or Jk or s or S due to the ag frequency. AABB had changed the requirements for reference labs to 2+ cells (based on these facilities having limited quantities of rare cells or antisera).

[Malcolm Needs ☆]

Hi David,

Yes, I can see what you mean.

We've had a couple of cases involving anti-Era in the last couple of years and, as far as I know, there are NO licensed anti-Era grouping reagents in the world. We had to use some SCARF derived antisera to group the two patients, using positive and negative controls, as there was no alternative.

Proving that there were no other underlying atypical alloantibodies was fun! Fortunately, we had 6 other examples of ABO compatible Er(a-) cells available to us, and were able to rule out everything apart from a potential anti-K using these and the patient's own typing, and giving K- blood was no problem.

[Cathy]

I agree with David, you may want to consider having an additional panel on hand. Using your screening cells in your rule outs or as one of your three positives/negatives may help but again, if your dealing with something other than a K or E you may have trouble. How are you doing your ruling out? We prefer to rule out cells with homozygous expression. If we have to use heterozygous we require two negatives, (when using gel or peg). When doing rule outs and using LISS as the testing media, we do not use cells with heterozygous expression.

[dawntr50]

I am new to this talk page and I am new to blood banking. I am training in BB right now and I dont understand how to identify multiple AB'S on panel cells. I am a seasoned tech as far as POL'S are concerned but now I am training in a hospital and I am loss in this area of this dept . I took BB 25 yrs ago during my apprenticeship and never saw it again. If some one could please tell me the proper steps to follow and the reason behind those steps in plain English I would appreciate your help. I am interested in learning to ID multiple AB's with panel cells from start to finish including how dosage applies. Please help if you can. Thanks

[Malcolm Needs ☆]

I'll give it a go, but I expect others will have more to say.

The normal 10 to 12 cell panel should enable you to identify common mixtures of antibodies, such as, for example, anti-E+K, anti-D+C+E, etc, but it is always worthwhile having more than one panel available to you, so that you have extra cells to identify more difficult mixtures.

It is also worthwhile using a papain-treated panel, as some antigens are destroyed by such treatment (M, N, Fy(a), Fy( and, usually, S and s). If, therefore, one or more antibodies in the mixture react by IAT with untreated cells, but not with papain-treated cells, then this is a guide that the antibody may be directed against one or more of the above antigens (although that short list is by no means comprehensive; there are many other antigens destroyed by such treatment, but the antibodies against these other antigens tend to be quite rare).

The next thing you can try, if you have the reagents available, is to fully phenotype the patient's red cells, as the patient will be unable to produce an atypical alloantibody directed against an antigen they themselves express. This, of course, also assumes that the patient has not been transfused within the previous 3 months (approximately) and does not have a positive direct antiglobulin test.

After this, it tends to become something of a lottery, depending upon the reagent sera and red cells that are available to you.

If you have sufficient cells, you could try adsorbing the patient's serum/plasma with red cells of known phenotype, and then a) testing the adsorbed plasma with the full panel and/or eluting the antibody(ies) from the cells used to perform the adsorption, and performing an antibody identification on this eluate.

Although I mentioned papain treatment of red cells above, there are many other enzymes that can be used, each of which (or, at least, most of which) "cut" amino acid residues at different places and will destroy different antigens. An extremely useful list can be found in the excellent book written by Marion Reid and Christine Lomas-Francis, "The Blood Group Antigen FactsBook", 2nd Edition, 2004, Academic Press, ISBN Number 0-12-586585-6, Library of Congress Catalog Number 2003102995.

If you think you have a mixture of IgG and IgM antibodies, you could try treating the patient's serum/plasma with dithiothreitol (DTT), which breaks the J-chains holding together the IgM molecules, leaving you with just the active IgG molecules. You then perform an indirect antiglobulin test using a monospecific anti-IgG reagent.

I suspect, however, that the average Hospital Blood Bank will not have all, or even many of these reagents available, and so I really would suggest that, if you get stuck, you send a (large) sample of the patient's blood to a Reference Laboratory; afterall, that is what they are there for!

Moving on to dosage, certain antibodies (usually weak examples) will react more strongly, or even only, with red cells apparently expressing homozygosity for a particular antigen (remember, the antigen itself is not homozygous, it is only the gene governing the expression of the antigen that can be homozygous). Commonly, you will find that an anti-M wil react more strongly, or only, with M+N- cells.

A similar pattern can often be seen with an anti-Fya and Fy(a+b-) red cells and with anti-Jka and Jk(a+b-) red cells, although, once again, this list is very far from comprehensive.

It is well to be aware of the fact that "apparent homozygosity" means exactly what it says. Unless the donor of the red cells in the panel has been genotyped for particular blood group loci, a red cell typing as Fy(a+b-) can, for example, actually be FYA/FY (the FY gene is silent) and so the cell may react with the same strength as an Fy(a+b+) red cell with a particular anti-Fya (they are, after a fashion, heterozygous, or even hemizygous, for the FYA gene, and will express fewer Fy(a) antigen sites on the red cell surface than a genuine FYA/FYA [Fy(a+b-)] red cell).

I hope this helps a little, but I am certain that others will be able to put the above in a simpler way, add to it, express different opinions, etc, but it is a start!

[Malcolm Needs ☆]

I forgot to say, incubation of the tests at various temperatures can also help to elucidate antibody specificities in some cases.

[Mabel Adams]

To add another angle to Malcom's post: the process of Ab identification starts with a process of elimination. You look at any cells on the panel which gave you a negative reaction. The logic is, if this sample contained an antibody to an antigen that is strongly expressed (i.e. double-dose, which means from a person homozygous for the gene coding for that antigen--but with the caveats above considered) then this cell would have reacted; therefore you can cross out any specificity at the top of the panel sheet for which the non-reactive cell is strongly postive. Proceed through your panel doing this for any non-reactive cells. If you have very few non-reactive cells, run additional cells--especially ones negative for high frequency antigens and those negative for the more common specificities like K, E, Fya in the hopes of getting a few more negative reactions that allow you to rule out more specificities. All this is assuming you have a negative DAT or auto control so you aren't chasing the nearly hopeless case of finding negative reactions in the presence of a warm auto antibody.

Once you have ruled out enough antibody specificities to have only a few left (at this point don't worry about those that have one or no positive cells on the whole panel) you need to choose cells specifically to help sort out those specificities. In a perfect world, you will find cells that are each positive for one of the remaining specificities but negative for all the others. Once you run these, you will be able to rule out more specificites.

The additional techniques above can also help. Antigen typing is most useful when the specificity is not too rare (i.e. it is less helpful with K than with Fya.)

I must quit or this will time out on me. Hope that helps some.

[GilTphoto]

We get a 16 cell panel every 2 weeks, so we have 2-3 in date panels at a time. We're not too quick to throw out the expired panels either, so we usually have 6 panels. Last month we had a patient with an anti-E, c, K, and Jkb. It took cells from all 6 panels to rule out others and rule in these 4 antibodies. Whatever was detected with expired cells, we will retest as new panels come in with appropriate cells.

Does anyone freeze rare cells from panels? I see Medion is selling some products for freezing and thawing aliquots. Does anyone have experience using it?

[Malcolm Needs ☆]

We freeze rare cells all the time (about 600 in our collection), but these are from our donors (I am at a Blood Centre).

We use something called glycigel. I'll be honest, although I am the Manager, I haven't a clue what this is! I'll find out from my colleague Alan Gray (who does all our rare screening) and then post again.

By the way, I meant to say that I agree with every single thing Mabel says.

Edited May 6, 2009 by Malcolm Needs
Forgot last sentence.

[ckcheng]

In addition to all the wonderful information provided above in guiding one to do antibody workup, I would like to add a little info in the usefulness of incorporate an autocontrol.

If autocontrol is positive, perform DAT.

If DAT is negative - probably positive reactions are due to the testing medium. Choose another testing method.

If DAT is positive - may be autoantibody(ies) +/- alloantibody(ies).

If autocontrol is negative, perform DAT is not necessary.

Most likely it is an alloantibody(ies). Or antibody against a high incidence antigen if all panel cells are reactive.

But do not forget to ask for patient history cuz they are very useful information when doing antibody workup.

CK Cheng, MSc, SBB(ASCP), CQA(ASQ)

Hong Kong

May 9, 2009

[Malcolm Needs ☆]

I agree with an awful lot of what CKCheng says, but I have certainly seen quite a few cases of auto negative, DAT positive, particularly in cases of either a delayed haemolytic transfusion reaction or a delayed serological transfusion reaction.

A DAT is not an onerous test to perform, and is always worth doing in such circumstances I think.

[Mabel Adams]

Malcolm, are your auto controls done in gel or tube? Gel is awfully good at picking these up--sometimes too good.

[Malcolm Needs ☆]

Hi Mabel,

They are done in gel, and I do agree with you that this is an awfully good method to pick these up - sometimes too good.

That having been said, however, it is not that unusual for us to test a sample that shows a pan-agglutinin with papain-treated red cells in gel and, for example, an apparent allo-anti-E by gel IAT with a negative auto by IAT, but then to geta positive DAT and, after performing an eluate, finding that the eluate reacts more strongly with E+ red cells by gel, but still react with E- red cells.

Occasionally, very occasionally, we will then go on to alloadsorb the plasma and the apparent anti-E will be adsorbed to extinction by E- red cells. This is a bit of an esoteric test, and we only do it when we are short of work and bored!

To be honest though, as these patients are, by definition in this example, E- themselves, we wouldn't bother, and just recommend that E- blood be given (on the grounds that they are often transfusion dependent, or will become so, and we don't want them to form a real allo-anti-E.

[Malcolm Needs ☆]

I am new to this talk page and I am new to blood banking. I am training in BB right now and I dont understand how to identify multiple AB'S on panel cells. I am a seasoned tech as far as POL'S are concerned but now I am training in a hospital and I am loss in this area of this dept . I took BB 25 yrs ago during my apprenticeship and never saw it again. If some one could please tell me the proper steps to follow and the reason behind those steps in plain English I would appreciate your help. I am interested in learning to ID multiple AB's with panel cells from start to finish including how dosage applies. Please help if you can. Thanks

Hi dawntr50,

In Hot Topics you will find a thread named Antibody Identification Algorithm. This was aimed at your post, but I could not for the life of me remember where I had seen your post (I've now, obviously, found it).

The algorithm may be of absolutely no use to you whatsoever, but it's there if you haven't seen it.

[mrstymt]

i'm not sure if this fits - but since it's a thread on Antibody ID, i figured i'd ask.

i read in the AABB technical manual that Fisher's Rule of Three doesn't necessarily apply to all approaches in ensuring an identification with minimal statistical error (P<0.05), and that sometimes other methods are applicable (i.e. 2 negatives, 3 positives or 1 postive and 7 negatives and such...)

we had a case where a cord blood was DAT positive, but since we had barely any cord red cells, we recovered a very small amount of eluate that, if pipetted as per manufacturer's recommendations, we would only have enough eluate to last 9 cells in one panel by gel.

the mom's screen was anti-D, possible due to RhIg. if the panel's pattern matches anti-D up to cell 9 (which it did) - would the alternate approaches (non-Fisher's) be enough to confirm that the eluate demonstrated anti-D?

the reason why i'm asking is because, even though i suggested this information on alternate "rules", our BB techs have been hesitant in just ruling out anti-K with 1 heterozygous cell, regardless of its phenotypic frequency. instead, they have to find 2 additional selected cells at most to make sure it reacts negatively with the patient's serum or eluate. also, we usually would need 2 additional selected cells treated with ficin to rule out anti-C and anti-E by gel because there are no homozygous expression for those antigens with the D antigen being negative.

i was always taught that you use a combination of rule-outs with homozygous cells and the statistical "Rule of Three" if a pattern in reactivity matches that of an antibody in the cell lines. but it seems like at my institution, we're doing endless repeat selected cells trying to rule out anti-K, C, and E just because of the "Rule of Three", even if the reactivity pattern fits and at least one or two of the alternate "rules" apply.

thanks!

[Malcolm Needs ☆]

In the UK, our Guidelines (issued by the British Council for Standards in Haematology) only require us to have two positive reactions with a particular antigen and two negatives with a particular antigen (say 2 x S+s- and 2 x S-s+ to state that an anti-S is present), and to show that the patient is capable of making the antibody (in the example given, the patient must be S-s+, or, of course, S-s-). We haven't come across too many cases, if any, where this approach has not been effective.

Sometimes, of course, it is not possible to find two examples of red cells that are negative for a particular antigen (try finding two examples of Co:-3, for example - not easy!), and even if you do, you cannot necessarily exclude the presence of other clinically significant atypical alloantibodies, so for such cases it's off to the International Blood Group Reference Laboratory for our samples!

I disagree with you, however, mrstymt, that there are no C+,c-, D-, E+, e- red cells around; they are ryry. They are just incredibly rare (but they do exist). However, is it worthwhile looking for the presence of such antibodies (except, possibly in pregnant women)? Surely you would just give rr blood?

If you really want to identify such antibodies, you could always perform alloadsorption studies. We would, as a Reference Centre, but for most hospitals, this would not be an option.

Edited May 28, 2009 by Malcolm Needs
Forgot something.