Does anyone have experience with using techniques like adding extra plasma or incubating at 4 degrees for ABO testing in gel? I mean simple things like weak or missing reverse antibodies at a small hospital that does only gel testing so can't go back to tube.

I'm afraid not Mabel.  We always resort to tube, except if we think that the person has a cold auto-agglutinin that we can dissociate by warm washing with 37oC saline (but this is rarely sufficient)..

Having said that, of course, it may be that our hospital colleagues do this kind of thing, and only send us the one's that they cannot resolve by these methods, so we never actually see them.

I have not do this. But if this kind of doing make the reaction strength stronger , I will try.

We have never done that as it would change the cell-serum ratio that would not be optimum for reaction in gel. Like Malcolm says tube method is what we do resort to.

I presented a poster at the 1997 AABB convention regarding  26 ABO Reverse Grouping discrepancies detected in Gel when testing over 3500 patient blood samples.  An immediate-spin tube test (reverse grouping) resolved 19/26 discrepancies due to patient's anti-A/anti-B not detected in gel.  The immediate-spin tube test was 3-4+.  Expected anti-A or anti-B was not detected in gel or tube for 3 blood samples and remained unresolved even with extended RT incubation and cold incubation.

 

We also found that unexpected agglutination in the ABO reverse grouping test was sometimes resolved by centrifuging the blood sample for a total of 9 minutes (three times for 3 minutes) in a STAT-Spin express centrifuge (4400g) and then retesting in Gel.

We use tube to resolve ABO discrepancy.

Dear all, it is not that unusual to get weak or neg results in the reverse group in gel.  there are a number of things that you can do to force the reaction.

1.  Incubate at room temperature for 10-15 minutes before centrifuging.  If that does not work ->

2.  Put everything in the fridge for 30-60 minutes before pipetting (that means cards, cells and plasma).  Then pipette the cold red cells followed by the cold plasma into the cold card and put it back into the fridge for 15 - 60 mins before centrifuging.  There are three things that are really important here if you do this.  Firstly you absolutely must use at least 1 group O cell as well to rule out the possibility that any positive reactions you are seeing are due to cold antibodies. Secondly, when you pipette your cells into the reaction chamber, you absolutely must have an air gap between the top of the supernatant and the cells before pipetting the plasma on the top.  Otherwise, you may well get a 'double population' due to some cells starting to descend down the gel before the centrifugation process starts and before they've had a chance to react with these weak antibodies.  This is important due to the extended incubation.  Thirdly you absolutely must include controls.

3.  If that does not work, you can do the same thing, but use 100ul of plasma instead of 50ul.

4.  If that does not work, take an aliquot of your reagent red cells (including the all-important O cells, spin them down and remove the supernatant.  Replace with your normal LISS solution.  Repeat the test with these cells.  All the provisos for point 2 would also count for this option.

5.  If that does not work, you have no choice but to revert to tubes; but in my experience, the normal 'weak' iso-aggs due to old age or illness will be induced to stick one or two red cells together like this.

Hi Anna,

 

The only thing that I would question about your excellent and comprehensive answer is the use of LISS in point 4.

 

Many years ago, Sandy Holburn, the then Director of the Blood Group Reference Laboratory (it wasn't the International BGRL in those days!)  published a paper in which he stated that ABO antibodies agglutinate more weakly (not certain of my English grammar here!!!!!!!!!!!) in LISS than they do in NISS.  I was just wondering if NISS suspended red cells would be better, or whether modern LISS has overcome this problem?

 

Great post though.

 

Malcolm

Thanks, Anna, for all of that great information.  While on the topic, does gel reverse typing pick up strong cold reactive anti-M antibodies about the same as tube testing or more often or less?  

 

Also, you mention the air bubble being required in this case and it made me wonder if in IgG testing in Diamed gel you recommend having the air gap also or if it is more important in this testing than usual.  Ortho has always been ambivalent about the air gap--first training us that it was a good idea and then saying it wasn't really required.  Someone told me it makes gel AHG testing more sensitive.  I know I am redirecting this thread but don't want to miss this chance to pick your brain on whether we should strive to have the air gap in all gel testing or only in certain situations.

Mabel, we have many samples sent to us to resolve "ABO issues" (usually a group A that looks like it has an anti-A1) that turn out to be an anti-M that reacts with the reverse grouping cells.  It is almost worth thinking about having M-, N+ only reverse grouping cells!

Thanks, Anna, for all of that great information.  While on the topic, does gel reverse typing pick up strong cold reactive anti-M antibodies about the same as tube testing or more often or less?  

 

Also, you mention the air bubble being required in this case and it made me wonder if in IgG testing in Diamed gel you recommend having the air gap also or if it is more important in this testing than usual.  Ortho has always been ambivalent about the air gap--first training us that it was a good idea and then saying it wasn't really required.  Someone told me it makes gel AHG testing more sensitive.  I know I am redirecting this thread but don't want to miss this chance to pick your brain on whether we should strive to have the air gap in all gel testing or only in certain situations.

 

Hi Mabel,

It is very important to have air-gap in gel tube between the gel matrix and reaction chamber for reactants to mix and react. We use DIAMED gel and we were taught that order of addition is also very important - red cells first at an angle of 45 degree followed by serum/plasma added vertically (90 degree). This prevents showing 'dp' after centrifugation and also prevents inactivation of Coombs reagent by serum/plasma, as there is no washing step involved in gel.

Wow - lots of points here to answer.

Firstly Malcolm - I was not aware of the study you made.  I automatically said LISS because I was thinking about the LISS solution that goes with the cards you are using, and whose chemical parameters are fine-tuned to work with the buffers in the cards.  Ordinary saline can leave 'trails' in the gel sometimes because of incompatibility of the Osmolarity of the solution (Please don't ask me to go into any more detail than that anyone, that's about as far as my chemistry goes).

Secondly - antibodies like M and N in gel as compared to tube.  Well, I'm not actually aware of any study that has actually compared the rate of positives for these antibodies in tube as opposed to gel, but I know they are picked up, provided the cells have the required antigen and, as Malcolm said, can cause unexpected positive reactions in the reverse group.

Thirdly, the air-gap.  Aafrin is quite correct in what he (she?) says.  I would like to add a couple of things, though.  Firstly the air gap is important in that it allows the cells and the plasma to mix correctly in the reaction chamber, avoiding the situation where half of the red cells have never come into contact with the plasma before they hit the gel.  For an antibody that is of any reasonable strength, you probably won't see any difference in the reaction strength.  However VERY occasionally, when you have a very weak antibody, you can see a difference, and that could be a difference between a weak + reaction and a negative.  In some tests, like a one-step enzyme test (which I wouldn't recommend because it's so insensitive anyway) the difference will be bigger, because half of the cells won't even have come into contact with the enzyme.  The second thing is that the air-gap is much less important when you are pipetting with an Instrument (rather than pipetting manually with a pipette).  This is because the way the pipetting is carried out by the instruments is not quite the same. The plasma is pipetted with more force, so the mixing is more efficient.  This diminishes the need for the air gap.  I hope that answers everyone.

Thanks,Anna,for all of that great information.