DAT test

[momie]

Hi guys!

If someone could help me,

I was wondering why the washing RBC's step is excluded in the gel method Of DAT?

[John Eggington]

Firstly; remember that the AHG is incorporated into the gel column.

Cells are added to each column then undergo a specific centrifugation phase that draws cells through the column, but is too 'light' to have a significant affect on free immunoglobulin. So, during the centrifugation phase, red cells come in to contact with AHG, but the AHG is not neutralised, because it is not exposed to free immunoglobulin (only any immunoglobulin that happens to be bound to the red cells).

The same principle applies to the IAT, but with a prior incubation phase (maintaining an 'air gap' between the red cell/plasma mixture and the AHG impregnated gel column) to allow antibody and antigen to bind.

[momie]

Firstly; remember that the AHG is incorporated into the gel column.

Cells are added to each column then undergo a specific centrifugation phase that draws cells through the column, but is too 'light' to have a significant affect on free immunoglobulin. So, during the centrifugation phase, red cells come in to contact with AHG, but the AHG is not neutralised, because it is not exposed to free immunoglobulin (only any immunoglobulin that happens to be bound to the red cells).

The same principle applies to the IAT, but with a prior incubation phase (maintaining an 'air gap' between the red cell/plasma mixture and the AHG impregnated gel column) to allow antibody and antigen to bind.

thanks alot for answering...

[rravkin@aol.com]

Firstly; remember that the AHG is incorporated into the gel column.

Cells are added to each column then undergo a specific centrifugation phase that draws cells through the column, but is too 'light' to have a significant affect on free immunoglobulin. So, during the centrifugation phase, red cells come in to contact with AHG, but the AHG is not neutralised, because it is not exposed to free immunoglobulin (only any immunoglobulin that happens to be bound to the red cells).

The same principle applies to the IAT, but with a prior incubation phase (maintaining an 'air gap' between the red cell/plasma mixture and the AHG impregnated gel column) to allow antibody and antigen to bind.

John,

If I am understanding your explaination correctly you are saying that the low concentration of rbc's and the migration of the rbc's through the gel column itself act to effect the rbc's in the same way that washing would. The only problem is that with a 4+ reaction the rbc's do not migrate through the gel colomn at all. I have used the gel cards manully for DAT testing and have washed the rbc's prior to making the 0.8% rbc solution. In an automated testing system I think that the making of the 0.8% rbc solution may have the same effect as manual washing but I am not sure. Can you please explain further and thank you in advance.

[galvania]

Dear rravkin

I'm not John, but I am answering this point. I am sure John is not saying that the migration of the cells through the gel is the same as washing. If you think about what happens in a tube Coombs (direct or indirect), you have red cells at the bottom of the tube and plasma sitting on top of it. this means that free immunoglobulins are in the top layer of the mixture. This means that if you don't wash, the first thing the AHG comes in contact with is the free immunoglobulin, because you have to add the AHG from the top. In the gel system the whole situation is reversed. You pipette in your cells followed by the plasma. In exactly the same way as for tubes, free immunoglobulins remain in the top layer. The difference is that the cells come into contact with the AHG from the bottom, at the start of centrifugation. So the AHG comes into contact with sensitised cells BEFORE if can come into contact with any free immunoglobulins. So the free immunoglobulins don't get a chance to neutralise the AHG. When you get a 4+ reaction, it simply means that there were a huge quantity of strongly sensitised cells; the resulting agglutinates were so large that they could not descend into the gel matrix - don't forget that the small amount of supernatant above the gel line contains antibody, so the cells agglutinate here first.

Hope this helps

Anna

[John Eggington]

Anna has answered this far better than I would have.

There isn't really any equivalent to the 'wash phase', of the tube technique, in a coloumn agglutination test (which is the point of the test, I suppose!). Some people may 'wash' pateint red cells before performing a DAT by column technique, but this is nothing to do with the 'wash phase' requirements of the tube technique.

[rravkin@aol.com]

Dear rravkin

I'm not John, but I am answering this point. I am sure John is not saying that the migration of the cells through the gel is the same as washing. If you think about what happens in a tube Coombs (direct or indirect), you have red cells at the bottom of the tube and plasma sitting on top of it. this means that free immunoglobulins are in the top layer of the mixture. This means that if you don't wash, the first thing the AHG comes in contact with is the free immunoglobulin, because you have to add the AHG from the top. In the gel system the whole situation is reversed. You pipette in your cells followed by the plasma. In exactly the same way as for tubes, free immunoglobulins remain in the top layer. The difference is that the cells come into contact with the AHG from the bottom, at the start of centrifugation. So the AHG comes into contact with sensitised cells BEFORE if can come into contact with any free immunoglobulins. So the free immunoglobulins don't get a chance to neutralise the AHG. When you get a 4+ reaction, it simply means that there were a huge quantity of strongly sensitised cells; the resulting agglutinates were so large that they could not descend into the gel matrix - don't forget that the small amount of supernatant above the gel line contains antibody, so the cells agglutinate here first.

Hope this helps

Anna

Anna,

Thank you for your post it does help. However, I thought that washing the rbc's also caused losely bound proteins to be removed from the surface of the rbc's and I wonder if that is possible here in Gel. Additionally, when performing a DAT using the Gel colomn card we only add 50ml of the 0.8% rbc solution to the column. There is no top layer of further reaction volume solution. So if the cells are not washed and the 0.8% rbc solution is added then any bound or unbound proteins would remain homogenious in this reaction volume causing the potential for a false negative result despite the seperation of rbc's from the reaction volume via centrugation. I have been thinking that when the DAT is run using the Gel card and automation that a washing-like effect may occur when the 0.8% rbc solution is made. I appreciate in advance any further insight.

[galvania]

Hi rravkin

Yes, I see your point. In fact, I know from experience that some people routinely wash all weak positives before making the suspension to put them on to the gel in order to get rid of non specific reactions due to excess 'non specifically' bound immunoglobulins. If they are then negative, they report a negative DAT. However this practice has to be used with care, because low-affinity antibodies can also be detached from the red cells during the wash phase. I think a lot depends on WHY you are doing the DAT in the first place. In my opinion, people do far too many DATs. It is often done routinely with every sample - and then you pick up all sorts of clinically irrelevant positives. I am a firm believer that a DAT should ONLY be carried out where there are good reasons for doing one, and then that would get rid of far more irrelevant weak positives.

But I digress! When you do a DAT in the gel technique, it is true that you only have unwashed red cells suspended in your Diluent. However, you should be taking these cells from the red cell sediment, so the quantity of free plasma around will be minimal. In over 20 years of working with the gel technique I have never once heard anyone complain that they have had a false negative DAT in gel. On the other hand, I often hear that it is 'too sensitive'.

[John Eggington]

Agaiin, I agree with Anna. 'False negative' DAT is not something I'd associate with column technique (in an untransfused patient). You can sometimes get a stronger reaction if you add patient plasma to your DAT (and incubate like an IAT), which could relate to 'low affinity' auotantibody.

[rravkin@aol.com]

Hi rravkin

Yes, I see your point. In fact, I know from experience that some people routinely wash all weak positives before making the suspension to put them on to the gel in order to get rid of non specific reactions due to excess 'non specifically' bound immunoglobulins. If they are then negative, they report a negative DAT. However this practice has to be used with care, because low-affinity antibodies can also be detached from the red cells during the wash phase. I think a lot depends on WHY you are doing the DAT in the first place. In my opinion, people do far too many DATs. It is often done routinely with every sample - and then you pick up all sorts of clinically irrelevant positives. I am a firm believer that a DAT should ONLY be carried out where there are good reasons for doing one, and then that would get rid of far more irrelevant weak positives.

But I digress! When you do a DAT in the gel technique, it is true that you only have unwashed red cells suspended in your Diluent. However, you should be taking these cells from the red cell sediment, so the quantity of free plasma around will be minimal. In over 20 years of working with the gel technique I have never once heard anyone complain that they have had a false negative DAT in gel. On the other hand, I often hear that it is 'too sensitive'.

Anna,

Thank you once again for your time and explanation, and I if understand you correctly the rbc's are not washed when using the gel card with automation and/or manual practice. But I have to ask how a false negative DAT would be caught in the first place? It seems that in practice once the DAT is interpreted as negative no further work-up is performed on that specimen. Then if the DAT is actually positive, a certain amount of in-vivo hemolysis would occur and the patient's physician would note a decrease in hgb and perhaps order another DAT to be performed using the same methods. And it may not be caught again or if the DAT does come up positive we would certainly interpret that as a potentially delayed reaction and still not be compelled to revisit the initial specimen and perform the DAT using a different method. I would further say that the use of rbc sediment is routine but does not garantee, without washing, that they are free of bound proteins despite the separation from the plasma and the lesser percentage of plasma present in the aliquote used. Once again I look forward to your insight and thank you for your posts and your time.

Edited May 15, 2011 by rravkin@aol.com

[galvania]

Dear rravkin

Again I can see why you are worried, but I can assure you that, if you carry out the test according to the method in the box insert, you will not get false negatives. The answer is a very pragmatic one - the test was validated in this way with thousands of samples. If you think about it, the amount of free (rather than cell-bound) Ig would be tiny, and the Coombs reagent in the gel is extremely strong. So even if there were a minute amount of neutralisation due to the tiny amount of free IgG left in the cell sediment, then it would not be enough to give you a false negative. I agree that no, you would not see a false negative because you would not then confirm the wash-phase with IgG-coated cells, as there IS no wash phase to control. But let us imagine that you did the test, and it was negative. There were clinical signs of haemolysis. In that case, you would repeat the test; it if were still negative and there were real signs of immune haemolysis, then I would repeat the test using a number of different techniques and reagents. This is, however, a purely hypothetical situation. The only time in my life I ever saw this was many many years ago (pre-gel) when the patient had an AIHA with IgA only and we had to devise a very complex method involving immunofluorescence at the time to detect it.

[Mabel Adams]

I was taught that the free proteins (antibodies etc.) in the sample are not heavy enough to be spun down into the gel like the RBCs are. Gel testing uses a rather slow spin. If you could spin antibodies to the bottom of a serum or plasma tube, this would happen in all of our sample tubes whenever we centrifuge a specimen to get the plasma off of the cells! In fact the antibody might all be down amongst the cells in the bit of plasma that is in the sediment. We obviously don't expect that to be a problem. My, wouldn't the chemistry dept. have a hard time doing globulin and protein levels?

[Malcolm Needs ☆]

That's right Mabel. You would need a Svedberg centrifuge to do that.

[rravkin@aol.com]

Dear rravkin

Again I can see why you are worried, but I can assure you that, if you carry out the test according to the method in the box insert, you will not get false negatives. The answer is a very pragmatic one - the test was validated in this way with thousands of samples. If you think about it, the amount of free (rather than cell-bound) Ig would be tiny, and the Coombs reagent in the gel is extremely strong. So even if there were a minute amount of neutralisation due to the tiny amount of free IgG left in the cell sediment, then it would not be enough to give you a false negative. I agree that no, you would not see a false negative because you would not then confirm the wash-phase with IgG-coated cells, as there IS no wash phase to control. But let us imagine that you did the test, and it was negative. There were clinical signs of haemolysis. In that case, you would repeat the test; it if were still negative and there were real signs of immune haemolysis, then I would repeat the test using a number of different techniques and reagents. This is, however, a purely hypothetical situation. The only time in my life I ever saw this was many many years ago (pre-gel) when the patient had an AIHA with IgA only and we had to devise a very complex method involving immunofluorescence at the time to detect it.

Anna,

Thank you again for your insight but I am also concerned with potential steric hinderence caused by adsorbed plasma proteins other then IgG; most notably Wartins's Jelly when performing a DAT on Cord blood, that may result in a false negative reaction. I appreciate any further insight.

[galvania]

Dear rravkin

When you have Wharton's jelly in a cord blood sample, the sample MUST be washed at least 3 times before performing the DAT. This is the only time where it is obligatory to wash before testing. The problem, however, is not with false negatives, but that uncoated cells cannot get to the bottom on the gel because the Wharton's jelly 'sticks' them to the gel beads

[Stoogiesfreak]

A little differecnt twist on DAT testing.

We have a physician that orders a DAT on his patients post transfusion. We have tried to get him to be sure and place the order pre-transfusion, but so far no luck.

When we get orders for DAT on his post transfusion patients we pull the pre-transfusion sample and perform the DAT on both samples. I am thinking that a weak positive may be missed on the post sample by the donor cells.

Help!

Thanks,

John

[L106]

John - Do you know why the Dr. is ordering the DAT test? He's not trying to find evidence of transfusion reactions, is he? (in which case it would be appropriate to use the patient's post-transfusion sample, of course.)

Donna

[Stoogiesfreak]

I don't think so - the nursing notes on the patients do not imply any kind of reaction process. The physician is an Oncologist with a tendency to think everyone has a hemolytic process! I am beginning to think this is more of an education issue that an actual reaction or in vivo process.

John

[L106]

Yeah, we have one of those Oncologists, too. Frequently orders a DAT on her patients. (I often wonder if once-upon-a-time she failed to promptly recognize a hemolytic anemia and got "burned", so she's not going to let that happen again???)

Donna

[Stoogiesfreak]

Donna,

You may be very correct! Sorry to hear you are facing the same issue.

John

[galvania]

The thing is, though, are these patients who have had a transfusion in the previous couple of weeks and now need another transfusion? If that is the case, then it would be a totally appropriate request, as newly forming antibodies may all be attatched to the transfused red cells, without leaving any free antibodies present in the plasma, so you could have a positive DAT with a negative antibody screen, but the antibody would need to be identified by carrying out an eluate before the patient was transfused again. If this is not the case, then it is not really necessary - but then neither is a pre-transfusion DAT, unless you want it as a base-line

[Stoogiesfreak]

Thanks Anna,

The one we have had seem to be random. If these people have been transfused before we do not have a record. One patient, however, has had multiple transfusions, but to date has no evidence of an antibody. Her antibody screening has remained negative. Your thoughts make sense and I can see that on the one patient with multiple transfusions. The others are random. I like to do a pre DAT as a baseline, but find the orders for this perplexing.

Thanks!

John