I would like to see how others perform/result DATs.

Currently we perform DATs with Polyspecific AHG (IgG and C3).

If it is negative we are done. If it is positive we test with Anti-IgG only.

Currently we report as either Negative or Positive.

Do you follow this same testing pattern? Are you using complement specific reagents?

Do you report as Negative or Positive or do you indicate what is bound (or not bound) to the red cells?

Thanks for the info.

We test Poly, if neg we are done.

If positive, we test both IgG and C3.

We report out 'specificity' ("Positive, IgG", "Positive, C3", or "Positive, IgG and C3")

We don't report out reaction strength. There may be some value to reaction strength...?

Linda F

As a donor center we test using Gel, poly, IgG and C3b. Overkill? Probably! But it's just easy to set them all up as we do our work-up. In our reports we list all four results.

We do the same as bbbirder, but I am thinking of not stocking the poly because we have to buy 10 vials, and running both the IgG and C3 when we do DATs on adults.

We do it exactly like bbbirder.

I just switched from poly to IgG and C3 separately -- IgG only for babies and both for adults.

What do you do if the negative control is positive, i.e. the cells are agglutinated before the reagent is even added? We report the results as invalid. Some of the docs don't like that; they want us to tell them if there is complement, IgG or both on the red cells. Sometimes, we can guess based on the comparative reaction strengths of the control and the tests but I hesitate to report out our "best guess". We have our medical director discuss the specific findings with the clinician if they persist on wanting a definite answer. I would be interested in hearing other approaches to this situation.

If the control is positive, then this is usually an indication that there are strong cold agglutinins present. These may or may not be clinically significant, and knowing if there is really something on the red cells at 37° can be important.

In such cases, it is usually enough to thoroughly wash the red cells with warm saline (at least 3 times, maybe more - depends on the strength of the reaction), before carrying out the Coombs test. If this doesn't get rid of the cold auto-antibody, then it is a good idea to take a repeat specimen which is kept warm (using a thermos flask with water at 40°) from the minute it is taken from the patient, then transferring the sample to a water bath (or placing it into a beaker full of water in an incubator) before washing with warm saline.

hope this helps

What reagents do you use?

Immucor, Ortho. etc

Thanks.

We use immucor.

We use anti-IgG gel cards and buffered gel with anti-C3b,-C3d. We use both for adults and cords. The Immucor complement check cells are always 3+s - 4+ in the gel card. I also run the patient cells neat in the buffered gel (0.8%) to make certain I am not seeing any non-specific agglutination (yes, that is my paranoia easer).

Do you find gel DATs to be a lot more sensitive than tube? We certainly pick up a lot of auto controls in manual gel but the sample tested by tube DAT (IgG) is negative. I would not be thrilled to do eluates on all those that are recently transfused unless it was going to provide me some pretty meaningful information. This is why I have hesitated to use gel IgG cards for DATs.

Do you find gel DATs to be a lot more sensitive than tube? We certainly pick up a lot of auto controls in manual gel but the sample tested by tube DAT (IgG) is negative. I would not be thrilled to do eluates on all those that are recently transfused unless it was going to provide me some pretty meaningful information. This is why I have hesitated to use gel IgG cards for DATs.

For the same reason we do not use gel for DATs. We run our panels/AC in gel and if AC is positive, run DAT by tube. We are not comparing apples to apples but ......Sometime if patient is recently transfused and DAT is negative we try to run GEL DAT or run eluate on Gel.

Mabel - I do not run an auto ct with my antibody screens. We only run one now if we are doing an identification. I am not doing any more elution studies than I have in the past, but then again, I am not performing an auto. It has been apparent with gel (from the time I've started using it) that it is significantly more sensitive than tubes - any reactivity of 2+ or weaker in gel is usually negative in tubes. Why anyone runs a more sensitive test and then verifies it with one significantly less sensitive does not make sense (to me). Why use the more sensitive method to begin with (rhetorical)? It seems that if the method you use demonstrates a detectable level of reactivity, it would be incumbent on you to follow up that detectioni. Anyway . . . my peds docs like the extra sensitivity found in gel - they thought we were missing too many +DATs.

We have made a decision to switch to IgG and C3 separately, both on adults, IgG only on babies.

I was reviewing the package insert for the Anti-C3 reagent (Immucor)and I was curious about the Quality Control. Of course we will use Complement Check Cells as directed in step 1 for negative tests. I was curious about the saline control mentioned in step 2.

It reads as follows:

"False-positive reactions in the direct antiglobulin test may be recognized by carrying out a control test...in which two drops of saline are added at step 3, instead of Anti-Human Globulin. The control will facilitate the recognition of aggregates caused by 'complete' (IgM) autoantibodies that have not dissociated during the washing phases or to other causes (such as spontaneous agglutination on centrifugation)."

Are you reporting out this control step? If the control is positive, do you attempt to repeat the DAT with additional washes?

Thanks!

We run a cell control with both Anti-IgG and Anti-C3 when doing DAT's, but these are non-reportable results. In our computer system, only interpretations are posted to the EMR, and the rest of the individual test results stay on the BB side.

We use poly as a screening test. If positive, we test with anti IgG and anti C3b,C3d. The result is posted as DAT poly negative, or DAT poly positive, DAT IgG positive/negative, DAT C3 positive/negative.

I have a question about the complement check cells for David. If I understand correctly, you use neutral cards, you put ?50ul of a suspension of patient's cells followed by ?25ul of anti-C3, then centrifuge. If negative, you add 50ul of diluted check cells then centrifuge again and look for a positive result. Is that the way you do it? Also I don't understand why you add the check cells. I always understood that the purpose of check cells was to control the wash phase - and there is no wash phase. Is this a requirement over there?

Confused from Europe.......

Good Question . . . why do I run the check cell? They let me know that my reagent is working. I run them in parallel with my test. On my gel card I run the patient twice and the ct twice. One set with reagent and one with diluent. Why? It makes me feel better about the anti-C reactivity/non-reactivity.

Dear David - OK - I think I get it. You are running the check cells as a single positive control with a batch, not adding them to every negative well. Explains my confusion. thanks