Hello everybody,

We can´t find out the antibody...

Pts blood group is A1D CcD.ee K-, Anti-AB 4+, Anti-A 4+, Anti-B neg, Auto neg., Anti-A1(lectin) 4+ Anti-H neg. (backward: 3+ with B-Cells, negative with 0-, A1-, A2-cells)

IAT, Papain and CaptureR screens are positive at 37°C/ 3+ with all cells, IAT screen at room temperature 4+ (gel column cards, BioRad and Grifols)

Antibody identification was done in IAT 37°C (3+ with all cells), DAT weak+

IAT screen with washed cells 3+

But... in xmatch with RBCs from bloodbags (PAGGS-M): A1D CcD.ee K-, A2D CcD.ee K- and 0D CcD.ee K- all negative in IAT 37°C /gel column cards

Can anybody help me out?

Thanks,

K.

 

 

 

 

I am unfamiliar with some of your terminology, but, as I understand what I do understand, what you have may well be an auto-anti-H (or an auto-anti-HI); I am fully appreciative that I may be wrong because of the unfamiliarity with your terminology.

I'm sorry. I can't speak (and write) english well. :-/

We had positive reactions with every cell in IAT, but none in IAT xmatches.

Patients plasma was negative with 0, A1 and A2- cells,3+ with B-cells in NaCl.

 

Oh, I'm sorry, I wasn't criticizing; after all, I cannot either speak or write a word of German!

If you were cross-matching group A red cells, whilst your panel cells were group O, it sounds very likely that the patient has an auto-anti-H.  The reason I say this is because the H antigen is very strongly expressed on group O red cells, but very weakly expressed on group A red cells, and your reactions are exactly what I would expect to see in such a case.

my first thought was anti-H too, but in crossmatches with group 0 we had negative results.

Difficult!

Is the incubation time at 37oC identical in the screen and panel to that used in the cross-match, and are the techniques used identical (by that I mean column agglutination, tube methods, microplate methods, etc)?

yes, same.

column agglutination, BioRad gel cards

37°C for 15 min. incubated

 

Thanks for that.  This has me stumped for now, BUT, I'll keep thinking!

This is a complicated case. Are the screening cells reacting very good ( I mean does it have neg reaction) with other patients' sample?

OK, so I presume you are  diluent 2 to make your crossmatch suspensions?  Well, Diluent 2 is not identical to the buffer used for the cell suspension in the screening cells.  so it may be that this patient is reacting against something in the red cell suspension buffer which is common to both biorad and Capture - although I have never seen one reacting that strongly before.

On the other hand, it certainly does look like a cold antibody.  Are you doing both screen and XM manually or on an instrument?  And how exactly are you preparing your blood bag segments?

 

Looks like an interfering substance to me.  Have you tried rinsing your screening cells with diluent (1x is usually sufficient), re-suspending those cells in diluent, then re-testing in gel to see if the reactions go away?

 

Panel cells are ok in quality control and with other patients. I tried to wash some BioRad cells 3x with NaCl and resuspended it in diluent 2.Same reactions. Segment preparation: 5µl of segment-rbcs, 500 µl of dil.2.

 

What do you do with the segments BEFORE you take the 5ul?

just open it with segment openers (sarstedt)

So you put them in a tube, but you don't centrifuge them.  Is that right?  If so, look at the cell pellet at the bottom of the well.  In my experience, not centrifuging the segment can mean that your suspension is weaker than it would be if you were using a spun patient sample (or the reagent red cells) because your 5ul is not 5ul of packed red cells.  when weak reactions are involved, a weak suspension can make a weak positive reaction look negative.

This could mean that actually the crossmatch results were falsely negative

Right,we just use the rbcs straight out of the segment. But in this case, the reactions with panel cells were 3+,so(even with a little less strong suspension) i would expect weak positive reactions at least. I did some tests with packed cells today-same results...negative in IAT crossmatch.

In that case, I strongly suspect that Anna's (galvania's) suggestion about an antibody to a bacteriacidal additive used to suspend the screening and antibody identification panel red cells is correct.  Way back, I experienced this myself, and even if the cells were washed about 4 to 6 times, I could still not get rid of the positive reactions.

I always used to suspend my red cells in Dil2, until I was told to stop it by one of my bosses on the grounds that it was too expensive.  After that, we saw it more and more, and, of course, it actually cost us more to sort out the problem, than it would have done if we had continued to use Dil2.  Bosses who wear suits, rather than white coats, always know best though!

Thank you everybody. It is great to have someone to ask for help in such cases.

:-)