We had a DAT last night that was positive ( mixed field really) at AHG but after the 5 min room temp incubation ( we use poly) to enhance complement, it was completely negative. I think that patient may have some rouleaux or some non-specific protein that is causing stickiness but does not really indicate a pos DAT. Anyone else have any experience with this?

Thanks.

Our reference lab SOP states that the AHG test must be read within 1 minute. The only indication for reading after a 5-minute incubation is to detect weakly-reacting complement. Our lab would interpret that your patient's DAT is positive for IgG only and negative for complement.

Ditto with Heather.

Okay, firstly I repeated it and found it to be negative. Secondly, why would true agglutination ever go away? NO matter what any SOP says, a truly agglutinated specimen will stay that way no matter how long it sits at room temp! The patient also has no other clincal or lab findings to correlate a pos DAT.

Hi LaraT23,

I'm afraid that I have to disagree with you. If the equilibrium constant of the reaction is more favourable to dissociation of the antibody to the antigen, than to association at a certain temperature, then agglutination will be reversed. This is how the heat elution at 56oC and the Lui elution technique, using very cold temperatures work.

I would totally agree with others who say that the tests should be read as soon as possible after completion.

Then explain the immediate spin crossmatch that is pos and then neg at 37. Saline replacements also work for inc. protein and a pos will dissipate after one or two replacements. The result was mixed field ish and started to come apart under the scope. I will just never believe that any patient test will always behave by the book!

I agree ... reactions do indeed 'go away' due to dissociation over time (or temperature!) and yes, patient is weakly positive with the Anti-IgG portion of your AHG(Poly) and negative for the Anti-Complement portion.

To check this 'theory' ... run them separately using Anti-IgG and Anti-Complement. Do note that sometimes the AHG(Poly) is positive when the monospecific reagents are negative ... it's impossible to 'calibrate' them to match up exactly.

Hmm, I see what you mean LaraT23.

I just wonder if there is a "cold" reacting antibody present (giving the immediate spin result), together with an antibody within the Lutheran Blood Group System (which can 1. look stringy and 2. be quite fragile).

Pure speculation on my part, of course.

Have you checked to see if the patient received any plasma expanders? Was the sample collected from an IV site? Try having the sample recollected.

LShirley makes a good point.

By the way, what was the patient's diagnosis? I suppose that they hadn't been given ALG or something similar?

Again, only speculation on my part.

AHG reagent is IgG antibodies have two legs only, the principle it can detect the IgG or complement on the red cells is when the two legs link two red cells together, we can see the agglutination. But when the time past or respin the IgG antibodies in the AHG reagent may dissociated from the cells and rejoin, two legs of an antibody joined ith one cell, then the agglutination is disappear.

This theory can't interprete the complement DAT testing.

We had a DAT last night that was positive ( mixed field really) at AHG but after the 5 min room temp incubation ( we use poly) to enhance complement, it was completely negative. I think that patient may have some rouleaux or some non-specific protein that is causing stickiness but does not really indicate a pos DAT. Anyone else have any experience with this?

Thanks.

Hi Lara!

Had I been in your place, I would've recorded the finding and given the test result as negative, keeping in mind that patient didn't have anything clinically suggestive. Some studies quote positive DAT to the tune of 10% of hospitalized patients without any significance.

Recording the finding (i.e. + and then negative over 5 min) will help you correlate similar findings in future.

Anyhow, what was the result with anti-IgG (AHG), if it was done at all?

Also, do you use only tube technique only or some other also? Gel can provide better understanding with mf type of reactions.

Ortho manufacturer insert, for DAT, has a NOTE: "The sensitiviity of complement / anti-complement reactions can be increased by incubation at room temperature for 5 to 10 minutes and recentrifugation. However, the results obtained following immediate centrifugation should not be ignored because anti-IgG reactions may be adversely affected by incubation." Rev. March 2005