ABO and Rh Typing Procedure


The ABO system is the most clinically significant blood group system for transfusion practice, because it is the only blood group system in which antibodies are consistently and predictably present in the serum of normal individuals whose red cells lack the antigen. ABO compatibility between donor and recipient is the foundation upon which all other pretransfusion testing rests.

The D(Rho) antigen is, after A and B, the most important red cell antigen in transfusion practice due to its potent antigenicity. Unlike the ABO system, however, individuals who lack the D antigen do NOT consistently and predictably have anti-D in their serum.

The ABO and Rh is determined for all patients who are candidates for transfusion, for all blood donors, for all prenatal patients, and for all potential organ recipients and donors.


If not part of compatibility testing, a 4-5 ml clotted tube from an adult or a 2-ml clotted tube from a pediatric patient is sufficient. Samples collected in EDTA or CPDA (CPDA-1) are also acceptable. ABO forward grouping may also be done on a sample from a segment on a donor unit. Manufacturerís directions state clotted and EDTA samples may be tested up to seven days after collection. Segments from donor units may be tested up to the expiration date of the unit.


  • Reagent Anti-A, Anti-B and Anti-A,B
  • Reagent A1 and B Cells
  • Reagent Anti-D
  • Rh (D) control
  • 12 x 75 mm tubes
  • test tube rack
  • marking pen
  • dispo pipettes
  • physiologic saline
  • serofuge
  • lighted agglutination viewer


  1. Verify that patient information on the sample matches information on the worksheet.
  2. Centrifuge the sample and remove the serum to a labeled tube.
  3. Label 8 tubes with the first 3 or 4 letters of the patientís last name/or first 3 or 4 numbers of accession # and line them up in this manner in your test tube rack:
    Patient's Serum a1 b      
    Patient's 3% (WC) Washed Cells A B A,B* D DCo**
  1. Add 2-3 drops of the patientís cells to the WC tube, and prepare a washed 3% suspension. (See WASHED 3% SUSPENSIONS.)
  2. Add 2 drops of patient serum to both the a1  and b tubes. This will be used for the serum confirmation, also known as the reverse grouping or backtyping.
  3. Add one drop of reagent anti-A to the A tube, one drop of reagent anti-B to the B tube, and one drop reagent anti-A,B to the A,B tube. (*Note: A number of labs no longer use anti A,B reagent unless they are confirming O donor blood)
  4. Add one drop reagent anti-D to the D tube, and one drop Rh control to the DCo tube. (**Note: A number of labs no longer use Rh control reagent unless the forward typing is AB positive)
  5. Add one drop of washed patient cells to:
    A tube
    B tube
    A,B tube
    D tube
    DCo tube
  6. Add one drop reagent A1 cells to the a1 tube, and one drop B cells to the b tube.
  7. Shake all 7 tubes gently to mix.
  8. Centrifuge tubes in the serofuge for the calibrated time for saline suspended cells. Add an balance tube for the 8th tube.
  9. Gently resuspend each cell button individually and examine for hemolysis or agglutination with the aid of the lighted agglutination viewer. Grade results neg to 4+. (See GRADING REACTIONS.) A mixed field (MF) may also be reported.
  10. Immediately record the results in the appropriate spaces on the worksheet.
  11. If the Rh test is negative, add a second drop of anti-D, then centrifuge and read again. 
  12. Discard all materials in the isolation trash containers.
  1. To correct any errors, draw a single line through the error, and write the corrected result in above or below the error, along with your initials.
  2. Use the recommended strength of cell suspension - a too heavy or too light suspension may result in a false negative.
  3. Do not warm the ABO reagents before performing the test. Warming may reverse weak agglutination and cause a false negative.
  4. If an antibody seems to be missing or weak in the reverse typing, add 2 additional drops of patient's serum and recentrifuge.  If still missing, decrease the temperature for 10 minutes and then centrifuge.
  5. Do not attempt to serum confirm newborns, except those who type as AB. These should be serum confirmed to rule out polyagglutination due to a strong positive DAT or Whartonís Jelly from a cord sample.


Note that if hemolysis is present instead of agglutination, this is also considered a positive result.

+ 0 + 0 + A
0 + + + 0 B
0 0 0 + + O
+ + + 0 0 AB

The results must match one of the above patterns. Any deviation from the pattern constitutes an ABO discrepancy and must be resolved before the results can be reported. If the discrepancy cannot be resolved, the blood type must be reported as UNKNOWN. (See Flow Chart for Resolving ABO Discrepancies)


Agglutination in the D tube with no agglutination in the DC tube is reported as Rh positive. It is possible the agglutination may not be seen until after the 37oC incubation. Lack of agglutination after a 37oC incubation is reported as Rh negative. True Rh positive results should be at least 2+.

There must be no agglutination in the DCo tube for the Rh test to be valid. If there is agglutination in the DC tube, the test must be repeated using a multiply-washed cell suspension and chemically modified anti-D or saline anti-D and a saline control.


AABB Technical Manual, current edition

Authored by:  Peggy Schroeder, Revised by Rose Dickinson                                    


Clinical Microbiology Syllabus