PRINCIPLE AND APPLICATIONS
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.
REAGENTS, EQUIPMENT, AND SUPPLIES:
- 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
- lighted agglutination viewer
- Verify that patient information on the sample
matches information on the worksheet.
- Centrifuge the sample and remove the serum to a
- 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 3% (WC) Washed Cells
- Add 2-3 drops of the
patientís cells to the WC
tube, and prepare a washed 3% suspension. (See
WASHED 3% SUSPENSIONS.)
- 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.
- 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)
- Add one drop
reagent anti-D to the D tube, and
drop Rh control to the DCo tube. (**Note: A number
of labs no longer use Rh control reagent unless the forward typing is AB
- Add one drop of washed patient cells to:
- Add one drop reagent A1 cells to the a1 tube, and
one drop B cells to the b tube.
- Shake all 7 tubes gently to mix.
- Centrifuge tubes in the serofuge for the calibrated
time for saline suspended cells. Add an balance
tube for the 8th tube.
- 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.
- Immediately record the results in the appropriate
spaces on the worksheet.
- If the Rh test is negative, add a second drop of
anti-D, then centrifuge and read again.
- Discard all materials in the isolation trash
NOTES AND PRECAUTIONS
- 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.
- Use the recommended strength of cell suspension - a
too heavy or too light suspension may result in a false negative.
- Do not warm the ABO reagents before performing the
test. Warming may reverse weak agglutination and cause a false negative.
- 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.
- 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
INTERPRETATION - ABO GROUPING
Note that if hemolysis is present instead of
agglutination, this is also considered a positive result.
|PT CELLS +
||PT CELLS +
||PT CELLS + ANTI-A,B
||PT SERUM + A1
||PT SERUM + B
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)
INTERPRETATION - Rh TYPING
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
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