Antibody Screening

PRINCIPLE AND APPLICATIONS

The patient's serum is tested for the presence of clinically significant antibodies using an indirect antiglobulin method. The serum is tested against unpooled Group O cells selected to possess the relevant blood group antigens.

The antibody screen is routinely performed as part of compatibility testing; it is also performed upon request on prenatal patients, on Rh immune globulin candidates, as part of an organ transplant workup, or on other patients for whom an "indirect Coombs" is requested.

SAMPLE

Since the patient's serum is tested, a 10 ml clotted (red top) tube is preferred. A 4 or 5 ml red top is acceptable; a 2 ml red top is acceptable from a newborn.

The sample should be tested when fresh; old or improperly stored samples may lose complement activity and lead to false negatives.

REAGENTS, EQUIPMENT, AND SUPPLIES

  • 12 x 75 mm test tubes
  • Lighted agglutination viewer
  • Plastic test tube holder
  • Reagent Screening Cells I & II, and III 
  • Indelible marking pen
  • PEG
  • Large bore dispo pipettes
  • Anti-Human Globulin (Coombs serum)
  • 37oC waterbath or heat block
  • Coombs Control Cells
  • Serofuge 
  • Wash bottle with physiologic saline

PROCEDURE

  1. Verify that patient information on the sample matches information on the worksheet.
  2. Centrifuge the sample and separate the serum to a labeled tube.
  3. Prepare a washed 3% cell suspension from the patient's cells. See WASHED 3% CELL SUSPENSIONS.
  4. Label 3 tubes
    Patient last name/or #, I
    Patient last name/or #, II
    Patient last name/or #, III
  5. Using a large bore pipette, add 2 drops of patient serum to all tubes. Hold the dropper at a consistent 45-degree angle.
  6.  Add one drop of Screening Cell I to tube I; add one drop Screening Cell II to the II tube; add one drop Screening Cell III to the III tube.
  7. Shake all tubes to mix and centrifuge at high speed the time appropriate for the saline spin calibration in the serofuge.
  8. Gently resuspend and examine macroscopically for agglutination using the lighted agglutination viewer.
  9. Record all negative reactions as Ø; grade positive reactions on a scale of w+ to 4+.
  10. Again holding the dropper at a consistent angle, add 2 drops of PEG to all tubes.
  11. Shake to mix and incubate at 37oC at least 15 but no more than 30 minutes. (NOTE: IF A WATERBATH IS USED FOR THE 37oC INCUBATION, THE INCUBATION TIME MAY BE REDUCED TO 10 MINUTES).
  12. Wash all tubes 4 times, decanting well after each wash, mixing the cell button well between washes, and blotting the last drop of saline after the final wash.
  13. Immediately add one drop AHG to each tube, shake to mix, and centrifuge the time appropriate for the Coombs spin calibration in the serofuge (usually the same as the saline calibration).
  14. Immediately resuspend gently and check macroscopically for agglutination using the lighted agglutination viewer.
  15. Read and record results as described above, along with your initials and the date, if not already recorded on the worksheet.
  16. Add one drop Coombs Control Cells to all negative reactions and centrifuge 15-30 seconds in the serofuge.
  17. Resuspend and macroscopically examine for agglutination. There must be agglutination in this step or the test is invalid. Record results.

INTERPRETATION

  • The lack of agglutination indicates the absence of antibodies to antigens on the reagent test cells, and the test is reported negative.
  • Agglutination in either Screening Cell tube prior to the addition of Coombs Control Cells indicates the presence of unexpected alloantibodies. The test is reported positive and further testing is necessary to identify the antibody(ies). See ANTIBODY IDENTIFICATION.
  • Agglutination in the autocontrol (AUTO) indicates coating of cells circulating in the patient. This may be due to an antibody to medications, autoantibody, passively transfused alloantibody, or alloantibody coating transfused cells in the patient. Perform a DAT and obtain the patient's medication list, diagnosis and transfusion history.

NOTES AND PRECAUTIONS

  1. Use of PEG requires a strict serum/PEG ratio. Also, add no more than 2 drops of serum when using PEG.
  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 at the end of the procedure. Reasons for negative results here include:
  • Inadequate washing of the cells in the Coombs phase. Residual serum neutralizes AHG.
  • A small fibrin clot has formed in the tube, neutralizing AHG. Be sure sample has thoroughly clotted.
  • Coombs reagent was omitted or is inactive.
  1. Weak or questionable reactions may be enhanced by:
  • Increasing the amount of serum used to 3-4 drops. PEG must be omitted, and the incubation time extended to 30 minutes.
  • Omitting PEG and adding albumin to the test mixture. Extend the incubation time to at least 20 minutes.
  • Using enzyme treated cells. See manufacturer's directions.
  • Repeat testing using PEG to the test mixture. See manufacturer's directions.
  1. Occasionally you may get a moderately strong reaction at room temperature that persists weakly in Coombs. This may not be clinically significant if it is due to complement binding at room temperature from a cold-reactive antibody. To determine if the reaction is due to complement binding, do a pre-warmed screen (SEE PREWARMED CROSSMATCH TECHNIQUE).

REFERENCES

AABB Technical Manual, current edition

matc\abscrn.lab

Author:  Peggy Schroeder, Rose Dickinson
 

Clinical Microbiology Syllabus