Opinion on antibody panel

[Lekota40]

I was recently in the position to have to review an antibody ID performed by a coworker. I disagreed with the determination and called our supervisor asking if she had looked at it and was told yes and pretty much dismissed my concern over thinking it was incorrect.

So if anyone has these 2 panels and can let me know what their opinion is that would be great.

Ortho Gel panel lot VRA131 and Immucor panocell-16 lot 24493

Reactions obtained on gel panel are as follows:

Cell # reaction

1 2+

2 3+

3 2+

4 0

5 3+

6 0

7 3+

8 2+

9 2+

10 0

11 2+

auto 2+

Reactions obtained on Immucor panel with PEG as enhanceent

Cell# reaction at AHG-all neg at IS

1 2+

2 2+

3 2+

4 0

5 1+

6 2+

7 2+

8 2+

9 1+

10 2+

11 2+

auto 0

The other thing that assisted me in thinking that I am correct in what I think the antibodies are was based on the patient's phenotype which was also performed along with this panel.

Patient is E+e+C-c+ Jka+Jkb+Fya-Fyb+ M+N+S+s+ K-

Looking forward to any sort of feedback.

Edited August 3, 2009 by Lekota40

[Malcolm Needs ☆]

Difficult if you haven't got access to the panel phenotypes!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Any way these could be attached?????

[Lekota40]

Well here's my attempt at uploading the antigram sheets.

[Malcolm Needs ☆]

Looking at the panel results, one cannot exclude a combination of anti-C+K+Fya.

The one thing that worries me a little is that panel 1 shows an auto in IAT that is negative, whilst panel 2 shows an auto in IAT that is positive.

Did you papain-treat the panel cells to destroy the (possible) anti-Fya/Fya reaction and enhance the (possible) anti-C/C reaction?

:confused::confused:

[Lekota40]

unfortunately we do not have any sort of enzyme techniques to use. The person working on this as directed by the supervisor was told to do autoabsorptions as the next step. Yes the auto is positive in gel and negative in PEG but this seems to occur a lot I've noticed with gel as if some other thing is causing the gel to have positive auto controls. The DAT was performed and was negative.

My conclusion was the same as yours. I dont have the autoabsorption results but the coworker used 2 cells on the same Immucor panel and then an expired panel. The expired panel showed reactivity and I'm told to ignore it cause it didnt absorb out and needed to be absorbed again. The current panel managed to obtain negative reactions on cells 1 and 2. Coworker also did a peg panel on an older panel and came up with negative cells from 10-16 which leads me to believe the plasma was omitted from the tubes so I'm not buying that the absorption was performed correctly.

[Malcolm Needs ☆]

unfortunately we do not have any sort of enzyme techniques to use. The person working on this as directed by the supervisor was told to do autoabsorptions as the next step. Yes the auto is positive in gel and negative in PEG but this seems to occur a lot I've noticed with gel as if some other thing is causing the gel to have positive auto controls. The DAT was performed and was negative.

My conclusion was the same as yours. I dont have the autoabsorption results but the coworker used 2 cells on the same Immucor panel and then an expired panel. The expired panel showed reactivity and I'm told to ignore it cause it didnt absorb out and needed to be absorbed again. The current panel managed to obtain negative reactions on cells 1 and 2. Coworker also did a peg panel on an older panel and came up with negative cells from 10-16 which leads me to believe the plasma was omitted from the tubes so I'm not buying that the absorption was performed correctly.

I'm surprised that your supervisor suggested an auto-adsorption. Apart from enzyme-treatment, I would have gone for alloadsorption, on the grounds that the antibodies "look allo".

[Lekota40]

Thank you for the agreeing that an auto adsorption was NOT the next step.

This was reported out as a warm auto and anti-K which is why I was totally uncomfortable reviewing it and saying it was fine as is. So today at work ought to be interesting because my "wrong" reasoning is supposedly going to be discussed with me. My concern was that the patient received Fya and C positive units do to them only calling it anti-K but the units fortunately were negative for all 3.

I questioned the warm auto as it clearly had obvious negative cells, which obviously fit a pattern that looks like they are all negative for cells neg for K C and Fya. I'm told that warm auto dont have to react with all the cells.

Anyway just have to continue to be a good tech and hopefully wont cause further problems with daring to question a supervisor.

[Malcolm Needs ☆]

Thank you for the agreeing that an auto adsorption was NOT the next step.

This was reported out as a warm auto and anti-K which is why I was totally uncomfortable reviewing it and saying it was fine as is. So today at work ought to be interesting because my "wrong" reasoning is supposedly going to be discussed with me. My concern was that the patient received Fya and C positive units do to them only calling it anti-K but the units fortunately were negative for all 3.

I questioned the warm auto as it clearly had obvious negative cells, which obviously fit a pattern that looks like they are all negative for cells neg for K C and Fya. I'm told that warm auto dont have to react with all the cells.

Anyway just have to continue to be a good tech and hopefully wont cause further problems with daring to question a supervisor.

My goodness, you are going to have a talking to!

If I were your supervisor, I would be talking to you too, only I would be congratulating you on your serological logic.

Don't loose your temper with your boss, but stick to your principles.

:eek::eek:

[L106]

Lekota40 -

It would be interesting to hear your supervisor's explanation of why the patient's Direct Antiglobulin Test was Negative if the patient had a warm autoantibody.

It is true that warm autoantibodies may not always react with all the panel cells. Sometimes the warm autoantibody mimics a specific pattern (ie: frequently Auto-Anti-e). However, we often find that if you catch the patient early in the development of or at the tail end of the autoimmune process (ie: when the autoantibody is relatively weak), it may not react with all the panel cells.

Why bother to phenotype the patient's red cells for all those different antigens if you're not going to use that information? (Given the results that the patient's red cells were negative for only 3 of the tested antigens, I would have jumped on that like a Junebug.)

Please don't pull a "Ha! Ha! Gotcha!!!!" on your supervisor.....He or she may save your XXX someday if you miss something and make a poor call. (None of us is beyond an error....Some of us just make more than others!)

[Malcolm Needs ☆]

Lekota40 -

It would be interesting to hear your supervisor's explanation of why the patient's Direct Antiglobulin Test was Negative if the patient had a warm autoantibody.

It is true that warm autoantibodies may not always react with all the panel cells. Sometimes the warm autoantibody mimics a specific pattern (ie: frequently Auto-Anti-e). However, we often find that if you catch the patient early in the development of or at the tail end of the autoimmune process (ie: when the autoantibody is relatively weak), it may not react with all the panel cells.

Why bother to phenotype the patient's red cells for all those different antigens if you're not going to use that information? (Given the results that the patient's red cells were negative for only 3 of the tested antigens, I would have jumped on that like a Junebug.)

Please don't pull a "Ha! Ha! Gotcha!!!!" on your supervisor.....He or she may save your XXX someday if you miss something and make a poor call. (None of us is beyond an error....Some of us just make more than others!)

I agree with everything you say L106 (especially the bit about us all making mistakes), but one should also ask why, if it is supposedly an auto-antibody reacting with some (but not all) panel cells by IAT, he or she (the supervisor) can rely on the cell typing when some of them must rely on an IAT typing (e.g. the Duffy typing)? Seems strange (= daft) to me.

[L106]

Malcolm - I'm confused. (It doesn't take much!!!) Since the patient's red cells had a Negative Direct Antiglobulin Test, the various antigen typings should be valid (assuming the patient has not been recently transfused.) Right? (Let me know if I am missing your point.)

I think my previous post may have been misleading, too. For example, we often see patients who have a 1+ Pos Direct Antiglobulin Test and the Eluate reacts 2+ with all the panel cells (Ie: warm autoantibody, no specificity identified) and the patient's plasma reacts with some but not all panel cells (ie: there's a little bit of the warm autoantibody in the patient's plasma, but it is so weak that it only reacts with a few panel cells here & there.)

I did not mean to imply that I thought Lekoda40's patient had a warm autoantibody in his plasma. (I do not think he does; I agree with your alloantibody identification, Malcolm.) I was just saying that Lakoda40's supervisor's statement that warm autoantibodies don't have to react with all the panel cells can be true.

Donna

[JOANBALONE]

I am surprised that no selected cells were tested. Does your SOP suggest running selected cells? I would have put the warm auto theory on the back burner when the DAT was tested and found to be negative. Perhaps some one out there has a good antibody ID sop that they can share?

JB

[Lekota40]

The explanation I received today-and she still insists I'm wrong and she's right is that the coworker ruled out C and Fya with the autoabsorption so it had to be a warm auto. She had him perform the autoabsorption on a panel which expired in May, which he had used as a rule out panel and obtained the same sort of results on the top half of the panel-i.e. reacted with C, Fya and K pos cells and was negative with the cells negative for all three. The bottom half of the panel had all negative results. Her interpretation of this was that it was the warm auto reacting weakly cause of the age of the panel.

I argued the point that it makes no sense that he shouldnt have obtained reactions on the bottom half of the panel and suggested he forgot to add the plasma. She says its impossible as he did the whole panel at the same time. Obviously she was not sitting there watching him put drops (or not putting drops) of plasma in each tube so he could have erred-it happens. So she has him test 2 cells positive for C and Fya on this aged panel with the auto absorbed cells and they are negative so she claims this ruled them out cause if indeed the alloantibodies were there it would still be positive. I said using that logic why wasnt a K positive cell tested as well since we're all agreeing K is there? The response was eh guess we should have but we didnt.

So yeah-I was questioned about recent panels I did which she didnt like my interpretation work up of as a result of this. Unfortunately for me I have 23 years of blood banking experience and I'm at the level that I dont need to reprove everything and dont get whigged out about certain "unexplained" reactions. When a patient is in the process of starting to develop a new antibody or has some other interference from drugs etc you are going to have things that arent always cut and dry. You go with what you know for sure and recommend things like phenotypically compatible/crossmatch compatible blood. She has about 7 years experience and is currently getting her SBB. So she thinks that makes her more knowledgable than me. In some respects I acknowledge that she has new info and I appreciate getting it but I dont appreciate questioning my work.

So guess if anyone else reading this novel can explain better than she did how I'm wrong I'm going with I'm right.

[Malcolm Needs ☆]

Malcolm - I'm confused. (It doesn't take much!!!) Since the patient's red cells had a Negative Direct Antiglobulin Test, the various antigen typings should be valid (assuming the patient has not been recently transfused.) Right? (Let me know if I am missing your point.)

I think my previous post may have been misleading, too. For example, we often see patients who have a 1+ Pos Direct Antiglobulin Test and the Eluate reacts 2+ with all the panel cells (Ie: warm autoantibody, no specificity identified) and the patient's plasma reacts with some but not all panel cells (ie: there's a little bit of the warm autoantibody in the patient's plasma, but it is so weak that it only reacts with a few panel cells here & there.)

I did not mean to imply that I thought Lekoda40's patient had a warm autoantibody in his plasma. (I do not think he does; I agree with your alloantibody identification, Malcolm.) I was just saying that Lakoda40's supervisor's statement that warm autoantibodies don't have to react with all the panel cells can be true.

Donna

Hi Donna,

I can see why you are confused, because re-reading my post, I could have been a bit less obscure!

It depends what technology is used to perform the typing. In my own Laboratory, we use DiaMed gel for almost all typing. You will note that the Ortho panel (which, I belive is gel, although I'm not entirely sure) gave a 2+ with the patient's own cells. If, therefore, this technology was used to group the patient with any reagents that required an IAT, the results would be unreliable. In such a case, just to be on the safe side, and with knowledge that some antigens may be weakened and others destroyed, we would treat the patient's red cells with chloroquine before grouping, just to be on the safe side.

I do totally agree with you that a weak auto-antibody can mimic specificities and that not all cells in the panel will necessarily react by IAT; we'll take that as a given. This is why, however, I feel that a panel of enzyme-treated red cells is essential in this case. A true alloanti-Fya will not react with these panel cells, but an auto-antibody mimicking an anti-Fya may (not will, but may). However, the whole thing is best cleared up by an alloadsorption study, and an auto-adsorption study only serves to muddy the waters.

As I say though, I do totally agree with you. A couple of times we have identified an apparently clear cut alloanti-S, but the anti-S was reacting with papain-treated red cells (not unknown, but sufficiently rare to make us suspicious). In both cases, using extremely sensitive serological methods, we were able to demonstrate that the antibody present was, in fact, a very weak auto-anti-U.

I do not think, however, indeed, I am certain, that Lekoda40's supervisor has not proved his/her case.

I hope that I have made myself a little clearer, but if my ramblings are still confusing you, please do not hesitate so to say, and I'll give it another whorl!

:)

[Malcolm Needs ☆]

The explanation I received today-and she still insists I'm wrong and she's right is that the coworker ruled out C and Fya with the autoabsorption so it had to be a warm auto. She had him perform the autoabsorption on a panel which expired in May, which he had used as a rule out panel and obtained the same sort of results on the top half of the panel-i.e. reacted with C, Fya and K pos cells and was negative with the cells negative for all three. The bottom half of the panel had all negative results. Her interpretation of this was that it was the warm auto reacting weakly cause of the age of the panel.

I argued the point that it makes no sense that he shouldnt have obtained reactions on the bottom half of the panel and suggested he forgot to add the plasma. She says its impossible as he did the whole panel at the same time. Obviously she was not sitting there watching him put drops (or not putting drops) of plasma in each tube so he could have erred-it happens. So she has him test 2 cells positive for C and Fya on this aged panel with the auto absorbed cells and they are negative so she claims this ruled them out cause if indeed the alloantibodies were there it would still be positive. I said using that logic why wasnt a K positive cell tested as well since we're all agreeing K is there? The response was eh guess we should have but we didnt.

So yeah-I was questioned about recent panels I did which she didnt like my interpretation work up of as a result of this. Unfortunately for me I have 23 years of blood banking experience and I'm at the level that I dont need to reprove everything and dont get whigged out about certain "unexplained" reactions. When a patient is in the process of starting to develop a new antibody or has some other interference from drugs etc you are going to have things that arent always cut and dry. You go with what you know for sure and recommend things like phenotypically compatible/crossmatch compatible blood. She has about 7 years experience and is currently getting her SBB. So she thinks that makes her more knowledgable than me. In some respects I acknowledge that she has new info and I appreciate getting it but I dont appreciate questioning my work.

So guess if anyone else reading this novel can explain better than she did how I'm wrong I'm going with I'm right.

I am entirely sympathetic with your views Lekota40.

a) I am glad that I don't have to work for this supervisor.

I hope that she never has to deal with me if I need a transfusion (she may think my anti-B is some kind of strange auto-antibody)

[L106]

Well, Lakoda40, chances are the patient will be back again........(The bad penny always seems to turn up again.) Then hopefully the pattern will be obviously clear-cut......Let us know if and when that happens.

[LaraT23]

My thoughts exactly, a warm auto that is picky about the cells it reacts to? Methinks something stinks! Enzyme and separating the phases is the only way to go. That really is my only bone to pick with the gel. The days of being able to separate immdiate spin versus AHG and the differences are gone. I suppose it is a good sacrifice, I.e. better sensisitivity, but the old methods do have their advantages. I would do Immediate spin and enzyme treat to see who leaves. Also, as far as phentyping, if the patient has been recently transfused, it will be tricky. Do a cell separation or send one out, if it has been at least 3 days. Then phenotype.

[BSIPHERD]

I think it's very dangerous to go directly to either auto or differential adsorption when there are negative cells on the panel. I didn't look at the panel really closely, but it seemed that all cells that were positive were C, K, and/or Fy(a) positive and the negative cells lacked all three. It might have needed a few more selected cells to "rule in" some of the antibodies (we like to have 3 cells positive and 3 cells negative - and you have the 3 cells negative - ideally we like to have 3 cells positive so we'd like 3 C+ cells that were Fy(a-) and K-, etc. - but we don't always reach that). And you have antigen typed the patient and the patient is negative for those antigens.

Adsorbing plasma with cells causes a little dilution of the antibodies and changes things a bit so it's particularly dangerous to use when you've got clear cut antibodies to try to rule out those antibodies.

We never go to warm or differenital adsorption unless we have also shown warm autoantibody in an eluate. We don't do an eluate everytime, but we do one before we call anything warm autoantibody.

If I were on a jury and the patient had a bad outcome, I'd have to find for the patient.

[Malcolm Needs ☆]

I think it's very dangerous to go directly to either auto or differential adsorption when there are negative cells on the panel. I didn't look at the panel really closely, but it seemed that all cells that were positive were C, K, and/or Fy(a) positive and the negative cells lacked all three. It might have needed a few more selected cells to "rule in" some of the antibodies (we like to have 3 cells positive and 3 cells negative - and you have the 3 cells negative - ideally we like to have 3 cells positive so we'd like 3 C+ cells that were Fy(a-) and K-, etc. - but we don't always reach that). And you have antigen typed the patient and the patient is negative for those antigens.

Adsorbing plasma with cells causes a little dilution of the antibodies and changes things a bit so it's particularly dangerous to use when you've got clear cut antibodies to try to rule out those antibodies.

We never go to warm or differenital adsorption unless we have also shown warm autoantibody in an eluate. We don't do an eluate everytime, but we do one before we call anything warm autoantibody.

If I were on a jury and the patient had a bad outcome, I'd have to find for the patient.

I would agree that, in this case, there are clear cut specificities, and that more cells could be used to rule in and rule out.

I would dispute, however, that performing differential alloadsorptions in a case where there are some negative reactions is dangerous. Yes, there is some dilution effect, but a few years ago a colleague of mine deliberately spiked a panagglutinin with a very weak anti-D, and then adsorbed the plasma 8 times with some rr red cells. At the end of this process, the panagglutinin had disappeared, but the anti-D could be readily detected, albeit the reactions were very slightly weaker.

The trick is to ensure that the cells used for adsorption are really, really packed, by centrifuging on high for about 10 minutes, and then removing the supernatant and the top layer of cells, and then centrifuging again for another couple of minutes and taking off any residual supernatant and the top layer of cells.

After this, the only fluid left to dilute the antibody is, for want of a better way of putting it, "interstitial fluid".

I am always more worried when there are NO negative reactions, because then you may be taking out an antibody directed against a high incidence antigen that may be lurking under the auto-antibody.

Actually, differential adsorption can be extremely useful when you know that there is no auto-antibody present, but there is a complex mixture of "common" alloantibodies and when there is an alloantibody present directed against a high incidence antigen of known specificity. This can be adsorbed out, leaving any other alloantibodies present in at least one of the plasma samples that have been adsorbed.

I have used this method on several occasions with specificities such as anti-U and anti-HrB.

[BSIPHERD]

Malcolm, I love reading your stuff. Thanks for your input.

I didn't mean I would never do a differential adsorption when there are negative cells, I just wouldn't go directly there as my next step in the situation given. I don't think there's enough evidence that there is an autoantibody there to make that the next step - and that's where I think it can be dangerous. Certainly differential adsorption can be used to separate antibodies if that's the purpose.

Certainly technique can make a big difference on the amount of dilution that occurs, but the characteristics of the particular antibody can also make a difference on how it's reactivity is affected by the adsorption.

Love these discussions!!

[pstroud45]

I agree that it looks like a C,Fya and K. I also see positive autos with gel. If the DAT is negative then you do not have an auto.

[Malcolm Needs ☆]

Malcolm, I love reading your stuff. Thanks for your input.

I didn't mean I would never do a differential adsorption when there are negative cells, I just wouldn't go directly there as my next step in the situation given. I don't think there's enough evidence that there is an autoantibody there to make that the next step - and that's where I think it can be dangerous. Certainly differential adsorption can be used to separate antibodies if that's the purpose.

Certainly technique can make a big difference on the amount of dilution that occurs, but the characteristics of the particular antibody can also make a difference on how it's reactivity is affected by the adsorption.

Love these discussions!!

Yes; firstly I would agree with you that, in this particular case, there is insufficient evidence (well, none actually) for the presence of an auto-antibody.

Secondly, I agree with you about the antibody characteristics making a difference.

Thirdly, I would agree with you that these discussions are wonderful, especially if one can keep a mind open to other people's opinions, which, it would seem, nearly every poster can do.

P.S. Thanks for the comment.

[jayinsat]

The explanation I received today-and she still insists I'm wrong and she's right is that the coworker ruled out C and Fya with the autoabsorption so it had to be a warm auto. She had him perform the autoabsorption on a panel which expired in May, which he had used as a rule out panel and obtained the same sort of results on the top half of the panel-i.e. reacted with C, Fya and K pos cells and was negative with the cells negative for all three. The bottom half of the panel had all negative results. Her interpretation of this was that it was the warm auto reacting weakly cause of the age of the panel.

I argued the point that it makes no sense that he shouldnt have obtained reactions on the bottom half of the panel and suggested he forgot to add the plasma. She says its impossible as he did the whole panel at the same time. Obviously she was not sitting there watching him put drops (or not putting drops) of plasma in each tube so he could have erred-it happens. So she has him test 2 cells positive for C and Fya on this aged panel with the auto absorbed cells and they are negative so she claims this ruled them out cause if indeed the alloantibodies were there it would still be positive. I said using that logic why wasnt a K positive cell tested as well since we're all agreeing K is there? The response was eh guess we should have but we didnt.

So yeah-I was questioned about recent panels I did which she didnt like my interpretation work up of as a result of this. Unfortunately for me I have 23 years of blood banking experience and I'm at the level that I dont need to reprove everything and dont get whigged out about certain "unexplained" reactions. When a patient is in the process of starting to develop a new antibody or has some other interference from drugs etc you are going to have things that arent always cut and dry. You go with what you know for sure and recommend things like phenotypically compatible/crossmatch compatible blood. She has about 7 years experience and is currently getting her SBB. So she thinks that makes her more knowledgable than me. In some respects I acknowledge that she has new info and I appreciate getting it but I dont appreciate questioning my work.

So guess if anyone else reading this novel can explain better than she did how I'm wrong I'm going with I'm right.

I sympathize with you. I am in a very similar situation and have had to learn to do what is right and question what is wrong with uber-tact. I routinely review antibody workups where the tech, having been taught by the supervisor, has gotten rid of positive reactions by switching to tube with 1 hour incubation WITHOUT ANY ENHANCEMENT and just call it a "cold antibody" Oh, btw, cold panels (mini or otherwise) are not performed here ! (How can you call a "cold" without demonstrating increased reactivity at RT or below and decreased reactivity at 37and AHG???) I come along and run select cells and identify one or more allo's, bring it to the super's attention, just to be told i'm wrong. All I can say is document, document, document and pray for your patients.

[Lekota40]

Well I do pray for the patients. Luckily I work at a blood center with this supervisor and she doesnt know what I advise the clients that send us antibody IDs. In the mentioned things I was questioned on I advised based on what I would do as a tech in a hospital that wanted to immediately transfuse a patient. I do by the way still work per diem at a hospital under a very knowledgable supervisor and she has no problem with my interpretations, which is why I'm pretty much more ****** off than anything cause I'm a good tech and I do care about results I turn out and dont appreciate being questioned about them.

[Abid]

Although this is an old case however I believe all threads should last forever.

I agree with Lekota’s boss in that the patient has auto antibody and anti-K. Maybe the boss did not explain her conclusion. I would say, some auto antibodies are so weak and would not be detected unless enchantments media is used. However when you do the usual autocontrol by gel you always incubate cells with serum in LISS. LISS enhances antibody attachment to antigen. Where in circulation there is no enhancement media only time works. I suggest two things to make sure a combination of positive autocontrol and negative DAT is a real autoantibody:

1. Do the autocontrol without enhancement media (tube technique). The auto control will be negative.

2. Re-do the panel with the elute from autoadsorption, all cells which became negative will be Positive again.

This only my thoughts.