DAT and Elution

Hello.

A sample that is sent to the Red Cell Reference Laboratory for red cell alloantibody identification would have a case history, where it would include the results of the tests that have been previously performed by the requesting hospital blood bank laboratory. Such tests include positive antibody screening or positive Direct Antiglobulin Test (DAT), where further investigation is needed.

A positive DAT may indicate that there are antibodies coating the RBCs. This is usually the case in patients suspected to have haemolytic transfusion reactions, haemolytic disease of the newborn or drug-induced haemolytic anaemia. In such cases, the Red Cell Reference Laboratory staff would have to perform a DAT test to double confirm the result and the strength of the graded reaction submitted by the requesting hospital blood bank laboratory. The stronger the DAT, the more likely it is to be clinically significant.

Test: DAT
Specimen: Blood collected in EDTA tube (purple cap). A clotted specimen collected in a plain tube (red cap) can result in a false positive reaction for the presence of complement due to in-vitro activation of the complement cascade.
DAT test is performed using the gel card technology, where 50 μl of the patient’s 0.8% red cell suspension is added to the microtube column in the ID-DiaMed LISS/Coombs gel card and spun at 910 rpm for 10 minutes. This gel card contains polyspecific AHG reagent to detect IgG and also contains anti-C3d to detect complement. If DAT were confirmed positive, next we would have to find out whether is it IgG antibody or complement C3d or both that is coating the RBCs. We would use an ID-DiaMed DC-Screening II gel card to differentiate the reaction and the steps are the same as the one above. This gel card contains monospecific AHG reagents such as anti-IgG and -C3d. If only C3d were present, an eluate is not likely to yield any useful information and should not be performed in most cases. If IgG were present, we would proceed to do an acid elution.

Elution allows the dissociation of the antibodies from the RBCs cell surface to allow for identification. Acid elution is a relatively quick and easy method and is most suitable for the detection of warm reactive antibody. The washed antibody-coated cells are mixed with a glycine acid solution at a pH of 3.0. The antigen-antibody bond is disrupted and the antibody is released into the acidic supernatant. The supernatant is separated and the pH is neutralised to allow for antibody identification.

The acid elution kit used is called DiaCidel Kit that consists of ready-to-use reagents offering an easy working procedure for the elution of most common antibodies.

Reagents

1. DiaCidel wash solution (concentrated) containing Glycine-NaCl buffer. The working solution can be prepared by diluting the concentrated wash solution with distilled water in 1:10 (i.e. 1ml concentrated wash solution and 9 ml distilled water).
2. DiaCidel elution solution containing a low pH glycine buffer solution with a colour indicator.
3. DiaCidel buffer solution containing Tris buffer with bovine albumin (1.2%).
Test Procedure

1. Wash the red cells which are DAT positive once with isotonic 0.9% saline solution. At least 1ml of packed cells are required.
2. Wash 1.0 of packed cells 4 times with DiaCidel working wash solution.
3. Decant completely after the last wash and keep part of supernatant to test for the presence of irregular antibodies.
4. Add to the 1ml of wash packed cells with 1.0 ml of DiaCidel elution solution. Mix well.
5. Centrifuge immediately for 1 minute at 3000 rpm.
6. Transfer the eluate into a clean tube.
7. Add 5 drops (250 μl) of DiaCidel Buffer Solution to the eluate and mix well. Observe the forming of a blue colour that indicates a neutral pH of 6.5-7.5 us reached. If the blue colour is not obtained, add more buffer 1 drop at a time while mixing.
8. Centrifuge the eluate for 1 minute at 3000 rpm to completely remove any residual cells.
9. Eluate is now ready for testing.

Perform the normal procedure used for antibody identification with the eluate. Use the supernatant solution kept form the last wash in parallel as a negative control.

When you perform the antibody identification with the eluate, you would run an 11-cell panel without including the own patient’s red cells. Confirm the presence of an antibody if the pattern of reactivity matches a pattern of antigen-positive cells. If your DAT is positive but after doing elution and running on the 11-cell panel, the result is negative for all common antibody, we would not proceed on with the investigation. This could be due to many underlying reasons such as hypergammaglobulinemia or drug reactions.

Some links to check out the gel cards and the elution kit that I was referring to:
http://www.diamed.com/product_detail.aspx?id=123&navvis=
http://www.diamed.com/product_detail.aspx?id=122&navvis=
http://www.diamed.com/product_detail.aspx?id=494&navvis=

Thank you.
Indah.