Direct Antiglobulin Test Procedure (DAT)

PRINCIPLE

The DAT detects red cells coated in vivo with immunoglobulin or complement.

Washed red cells from a patient or donor are tested directly with antihuman globulin reagents. The test is used to detect hemolytic disease of the newborn, autoimmune hemolytic anemia, sensitization of red cells due to drugs, investigation of transfusion reactions, and hemolysis due to ABO-incompatible platelet transfusions or ABO- mismatched organ transplants.

The DAT may be set up with only the polyspecific tube first, and then repeated with the monospecific reagents of the polyspecific test is positive. This is generally done when you have no indication what the results of the test will be. However, if you are expecting the DAT to be positive, it is permissible to set up tubes for the polyspecific and monospecific reagents at the same time and test them all together.

SAMPLE

A 4 or 7 ml EDTA (lavender top) tube is the sample of choice. A sample from a red top tube may be tested, but is less desirable, because complement may become attached to red cells in vitro when they are exposed to their own serum, especially at lower temperatures. If you get a positive result due to complement on a sample from a red top tube, obtain a lavender top tube and repeat the test.

REAGENTS, EQUIPMENT, AND SUPPLIES

  • 12 x 75 mm tubes
  • Polyspecific AHG (Coombs)
  • Dispo pipettes
  • Anti-IgG
  • Indelible marking pen
  • Anti-C3
  • Isotonic saline
  • Coombs Control Cells
  • Serofuge
  • Lighted agglutination viewer
  • Microscope

PROCEDURE – POLYSPECIFIC TEST

  1. Add 1-2 drops of patient cells to a tube labelled with the patient’s initials. These are cells directly from the EDTA tube - they do not have to be diluted at this point.
  2. Wash this tube three times with isotonic saline.
  3. After the third wash, prepare a 3% suspension from the washed cells.
  4. Label 2 tubes – POLY IS and POLY 5” (plus the patient’s initials)
  5. Add one drop of the washed 3% suspension to each tube.
  6. Wash these tubes one more time. When decanting, position the tubes so that the cell button is on top. This will prevent too many cells from being lost in the washing process.
  7. Drain well, and blot dry with a biowipe.
  8. Immediately add one drop Polyspecific Antihuman Globulin (also known as AHG or Coombs serum) to both tubes, and shake to mix.
  9. Allow the 5” tube to incubate at room temperature 5 minutes.
  10. Centrifuge the POLY IS tube for the time calibrated for the Coombs spin on the serofuge.
  11. Immediately resuspend gently and examine for agglutination using the lighted agglutination viewer.
  12. Grade and record results in the POLYSPECIFIC I.S. column of the worksheet.
  13. If positive, continue with MONOSPECIFIC TESTING below. It is not necessary to read the 5” tube if the IS tube is positive, and it is not necessary to examine it microscopically.
  14. If the POLY IS tube is negative, examine for agglutination under the microscope. Record results under the I.S. – MICR. column of the worksheet. If positive, continue with MONOSPECIFIC TESTING below.
  15. If still negative microscopically, add one drop of IgG-coated Coombs Control Cells (Check Cells) to the tube and centrifuge.
  16. Examine for agglutination. Agglutination should be present in this step, or the test is invalid. See NOTES and PRECAUTIONS, below.
  17. Record the check cell results on the worksheet in the I.S. – CCC column. A checkmark () is sufficient if agglutination of any degree is present – no need to grade.
  18. If the POLY-IS tube was negative through the microscopic reading, centrifuge the POLY-5” tube after its incubation period and repeat steps 11-17 above, recording results on the worksheet in the POLY-5” column.
  19. If there is no agglutination in any of the steps BEFORE addition of the check cells (CCC), the test is interpreted as NEGATIVE and no further work needs to be done.
  20. If there is agglutination in any of the above steps BEFORE addition of the check cells (CCC), continue with MONOSPECIFIC TESTING below.

PROCEDURE – MONOSPECIFIC TESTING

If the patient test shows agglutination in step 11, 14, or 18, repeat the test using monospecific reagents and a saline patient control:

  1. Add 1-2 drops of patient cells from the EDTA tube to a new labelled tube. You cannot continue using the previously-washed cells at this point. See NOTES AND PRECAUTIONS below.
  2. Wash this tube 3 times.
  3. While this tube is washing, label three more tubes with the patient’s initials and: IgG, C3, Saline
  4. After the third wash, prepare a 3% suspension from the washed cells.
  5. Add one drop of the 3% washed cells to each of the tubes labelled in step C above.
  6. Wash these three tubes one more time, decant well and blot the last drop of saline with a biowipe. Remember to position the cell button UP when decanting.
  7. Add one drop Anti-IgG to the IgG tube, 1 drop Anti-C3 to the C3 tube, and 1 drop saline to the saline tube.
  8. Centrifuge and read as before, recording results in the appropriate columns on the worksheet.
  9. If the IgG tube is negative, examine it microscopically. Record all results in the appropriate place on the worksheet.
  10. After the 5" incubation, read the C3tube and record the results on the appropriate place on the worksheet.
  11. Confirm any negative IgG reactions with the IgG-coated Coombs Control cells, and confirm any negative C3 reactions with C3-coated Coombs Control cells. No need to confirm negative saline reactions.

PROCEDURE – POLYSPECIFIC AND MONOSPECIFIC TESTING DONE AT THE SAME TIME

NOTE THAT THIS PROCEDURE IS DONE WHEN THE RESULTS OF THE DAT ARE EXPECTED TO BE POSITIVE.

  1. Add 1-2 drops of patient cells to a tube labelled with the patient’s initials. These are cells directly from the EDTA tube - they do not have to be diluted at this point.
  2. Wash this tube three times with isotonic saline.
  3. After the third wash, prepare a 3% suspension from the washed cells.
  4. Label 4 tubes – POLY, IgG, C3, and SALINE (along with the patient’s initials)
  5. Add one drop of the washed 3% suspension to each tube.
  6. Wash these tubes one more time. When decanting, position the tubes so that the cell button is on top.
  7. Drain well, and blot dry with a Kimwipe.
  8. Immediately add one drop Polyspecific Antihuman Globulin to the Poly tube, one drop anti-IgG to the IgG tube, one drop anti-C3 to the C3 tube, and one drop saline to the saline tube. Shake all tubes gently to mix.
  9. Centrifuge all tubes for the time calibrated for the Coombs spin on the serofuge.
  10. Immediately resuspend gently and examine for agglutination using the lighted agglutination viewer.
  11. Read, grade and record results in the appropriate columns of the worksheet.
  12. If the IgG tube is negative, examine it immediately microscopically and record the result on the worksheet.
  13. If the Polyspecific or C3 tube is negative, incubate 5 minutes at room temperature, centrifuge, and read again. Record results.
  14. If the Polyspecific or C3 tube is still negative after the 5 minute incubation, examine microscopically. Record results on the worksheet.
  15. Confirm all negative results with the appropriate Coombs Control Cells. Remember to use complement-coated cells for the C3 tube. Record the results of the check cells on the worksheet.

INTERPRETATION

  1. No agglutination indicates a negative DAT - nothing is coating the patient's cells in vivo.
  2. Agglutination with Polyspecific AHG indicates a positive DAT, due to either IgG antibodies or complement coating the patient's cells. To determine which it is, the monspecific testing must be done:

  • Agglutination with Anti-IgG indicates antibody (immunoglobulin) is coating the cells.
  • Agglutination with Anti-C3 indicates complement is coating the cells.
  • Both IgG and complement may be present.
  1. If the polyspecific tube is positive but all the monospecific tubes are negative, repeat the entire test with the polyspecific and monospecific reagents.
  2. Agglutination with saline indicates either spontaneous agglutination due to cells heavily coated with antibody, (frequently IgM), spontaneous agglutination due to Wharton's Jelly coating cord cells, or polyagglutinable cells due to T activation. The test is inconclusive and the results cannot be reported.
  3. If the patient has a positive DAT, obtain the medications list, diagnosis and transfusion history. In some circumstances it may be necessary to perform an eluate to identify the antibody coating the patient's cells.
NOTES AND PRECAUTIONS
  1. The washing step must proceed uninterrupted. A delay in washing may cause antibodies to be eluted off the cell and washed away.
  2. AHG must be added to the cells immediately following washing. Antibodies may elute from the cells if they are allowed to sit in saline without the addition of AHG.
  3. The Coombs Control Cells must be positive in the Polyspecific and IgG tubes at the end of the procedure. Reasons for negative check cells include:
  • Inadequate washing of the cells. Residual serum neutralizes the AHG so that it cannot react with the antibody-coated Coombs Control Cells at the end of the procedure. Be sure the cells are thoroughly resuspended between each wash.
  • A delay in adding AHG. Antibodies that were attached to the red cells may elute off into the saline, and neutralize the AHG when it is added. Add the AHG immediately after the final wash.
  • A small fibrin clot has formed in the tube, neutralizing the AHG. Be sure the sample has thoroughly clotted, if using a red-top tube.
  • AHG was omitted, is inactive, or was diluted out by too much residual saline in the tube. Drain the last wash well and blot before adding AHG.
  1. If the test is positive for complement and the sample was drawn into a red top tube, repeat the entire test on a lavender top tube. If the C3 is now negative, this indicates the previous positive result was due to in vitro binding of complement, and is a false positive.

REFERENCES:

AABB Technical Manual, 13th Edition, current edition

 

 

Clinical Microbiology Syllabus