34 Direct Antiglobulin Test (DAT)

Principle

The Antihuman globulin (AHG) method can demonstrate the presence of “in-vivo” sensitization of RCs with IgG and /or complement (C3b and/or C3d).

Specimen

Red cells from an EDTA-anticoagulated blood sample.

Reagents

1. Antihuman globulin (AHG) reagent: polyspecific (anti-IgG and anti-complement) and monospecific anti-IgG, & anti-complement (anti-C3b, -C3d).
2. IgG-coated red cells. ( Control Cells- CC’s)
3. Complement-coated red cells, if instructed by the manufacturer, as available.

Procedure

1. Label tubes with patient name and MRN or donor identification and the name of the AHG reagent being used (poly, anti-IgG or anti-C3d).
2. Place 1 drop of the 2-5% RC suspension into a clean, labeled test tube.(2 drops if older or weaker suspension as cells lost in washing procedure).
3. Wash 4 times with saline.
4. Completely decant the final wash and blot dry.
5. Immediately add 2 drops “straight up and down” of the appropriate antihuman globulin (see note 2) reagent and mix well.
6. Centrifuge for 20 seconds.
7. Gently suspend/ dis-lodge the cell button and examine macroscopically.
8. Grade and record results immediately while holding tubes in hand.
9. If using
   • AHG (polyspecific) or anti-complement (-C3b, -C3d), incubate non-reactive tests at room temperature for 5 minutes and then centrifuge for20 seconds.
   • Anti-C3b, C3d may take additional time to combine with sensitized RCs
   • Anti-IgG only, no second incubation/read is necessary.
10. Grade and record second results below the first result immediately while holding tubes in hand.
11. Control all negative polyspecific AHG or anti-IgG tests by adding 1 drop of IgG coated red cells (CCs) and all anti-complement antisera with complement-coated red cells (if instructed by manufacturer).
12. Mix and centrifuge for 20 seconds.
13. Examine for agglutination and record results immediately while holding tubes in hand.

Interpretation

1. The DAT is positive when agglutination is observed either after immediate centrifugation or after the centrifugation that followed room temperature incubation. IgG-coated red cells usually give immediate reactions and should not be ignored, because anti-IgG reactions may be adversely affected by incubation. Complement coating may be more easily demonstrated after incubation. Even though this is true, mono-specific reagents are required to determine if IgG ,Complement  or both is coating the RCs.
2. The DAT is negative when no agglutination is observed at either test phase, and the CCs are agglutinated. If there is no agglutination or the results are questionable, the test is invalid and must be repeated. Record the results of CC’s while tube is in hand(1-4+).

Notes 

1. Perform cord DAT’s using monospecific anti-IgG. It is not necessary to perform the DAT on a cord using polyspecific antihuman globulin reagent because only clinically significant IgG antibodies will cross the placenta.
2. DATs can be performed in Gel cards following manufacturers recommendations.