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ABO subgroups and discrepancies

17 min

Skills you'll build3 skills
  • Use reverse grouping, lectin tests, and history to explain a weak ABO forward reaction
  • Assign an adult ABO group from concordant forward and reverse results
  • Choose the next check for a discrepant ABO reaction pattern

At the bench: Resolve a blood group discrepancy before selecting compatible components

Try first

Try first

A 2-day-old's red cells react 3+ with anti-A, 0 with anti-B and 0 with anti-A₁ lectin from Dolichos biflorus. How is the subgroup reported?

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Forward and reverse grouping must agree

Forward grouping tests the patient's red cells with anti-A and anti-B. Reverse grouping tests the plasma with A1 and B reagent cells. CLIA requires both, and the results must agree before the laboratory records the group.1,3

ABO groupAnti-AAnti-BA1 cellsB cells
ONegativeNegativePositivePositive
APositiveNegativeNegativePositive
BNegativePositivePositiveNegative
ABPositivePositiveNegativeNegative

A young infant's anti-A and anti-B are unreliable, so the infant procedure generally assigns the group from forward grouping.3

Resolving ABO discrepancies

When forward and reverse grouping disagree, the weak, missing or unexpected reaction usually shows which side has the problem. Check patient identification, specimen labeling, reagents and technique first.3 The reaction pattern then guides the next step:2,3

  • A weak or missing reverse reaction fits a young infant, low immunoglobulin or immunosuppression. Review the history and extend the reverse grouping by the procedure.
  • A weak or mixed-field forward reaction fits a subgroup, a recent transfusion or a transplant. Review the transfusion and transplant history first.
  • An extra reverse reaction fits anti-A1, a cold antibody or rouleaux. Group O cells, A2 cells, an autocontrol and microscopic examination distinguish them. Saline replacement disperses rouleaux.
  • An extra forward reaction fits acquired B, red cells coated with autoantibody or plasma proteins, or an out-of-group transfusion or transplant. Review the diagnosis and the transfusion and transplant history. Repeat grouping with washed cells and the required reagent control. A direct antiglobulin test checks for immunoglobulin or complement coating.4

The group is recorded only after the discrepancy is resolved. Red cells needed before then are group O.2

Identifying A subgroups

A2 cells react with anti-A and stay nonreactive with the anti-A1 lectin from Dolichos biflorus. Some people with A2 make anti-A1, usually a cold antibody that shows as an extra reaction with A1 reagent cells. A newborn's weaker A expression can also leave the lectin nonreactive.3 Reverse grouping, lectin tests and the history are interpreted together before a subgroup is assigned.3

Follow the bench demonstration

Sources for this step
References
  1. Standard: Immunohematology, 42 CFR §493.1271 (2026). Accessed September 27, 2026. https://www.ecfr.gov/current/title-42/chapter-IV/subchapter-G/part-493/subpart-K/section-493.1271
  2. Association for the Advancement of Blood & Biotherapies. Fundamental Standards for Blood Collection and Transfusion. 2nd ed. AABB; 2025. Effective January 1, 2026. Accessed September 27, 2026.
  3. Bloch EM, Campbell-Lee S, McKenna DH Jr, Montemayor-Garcia C, Schwartz J, Shaz B, Storry J, eds. Technical Manual. 22nd ed. AABB; 2026.
  4. Rhees JR. Resolution of ABO Discrepancies. Lecture slides. ARUP Laboratories Institute for Learning; 2018. Accessed November 2, 2026.

Watch one

Read each decision and its explanation.

Walter Coulter, 84, a retired ferry captain with chronic lymphocytic leukemia and low immunoglobulin levels, has a type and screen before hip repair. His record from 2 years ago shows group A.

TestReaction
Anti-A, patient cells4+
Anti-B, patient cells0
A1 cells, patient plasma0
B cells, patient plasma0

What is the next check?

  1. Check the specimen label, the request, the reagents and the recorded reactions. All are in order.

    A wrong specimen or a missed reagent can produce any pattern, so identification and technique are checked first.

  2. The forward grouping reads group A. The reverse grouping, with no anti-B, reads group AB. The results disagree.

    Forward and reverse grouping must agree.

  3. The missing reaction is anti-B, so the problem is in the reverse grouping.

    The weak or missing reaction shows which side has the problem.

  4. His age, his leukemia and his low immunoglobulin all fit weak antibody production, and his record agrees with the forward grouping.

    History often explains a missing plasma antibody.

  5. Repeat the reverse grouping with extended room-temperature incubation under the procedure, with group O cells and an autocontrol.

    Longer incubation lets weak antibody react, and the controls show whether the plasma reacts with every cell.

  6. Record the group only when the reverse grouping supports it. Red cells needed before then are group O.

    A group recorded from one side can be wrong.

The missing anti-B points to low antibody production. The next check is an extended reverse grouping with group O cells and an autocontrol, and the ABO group is not recorded until that result is back.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

An adult's cells react with anti-A only. Plasma reacts with B cells only. What is the ABO group?

Incorrect. Group O cells lack both A and B antigens, and expected plasma reacts with both A₁ and B cells.

Correct. The cells express A antigen and the plasma contains expected anti-B.

Incorrect. Group B gives the opposite pattern: anti-B reactivity and expected anti-A in plasma.

Hint
  1. Forward grouping reads the antigens on the patient's cells.
  2. The plasma contains antibody to the A or B antigen the cells lack.

Review D typing

Problem 2 of 3 · Hints available

A 67-year-old with multiple myeloma has these results. Under the microscope, the cells in the A1 cell tube stack like coins.

TestReaction
Anti-A, patient cells4+
Anti-B, patient cells0
A1 cells, patient plasma2+
B cells, patient plasma4+
O cells, patient plasma2+
Autocontrol2+

What is the next check?

The reverse grouping does not yet support group A. The discrepancy is resolved before a group is recorded.

Assigned ABO from one side of a discrepancy

A weak subgroup, a mixed population after transfusion or transplant, or a missing plasma reaction can each make one side of ABO testing misleading. A group assigned from that side can be wrong and lead to incompatible components, which is why the discrepancy is resolved before assignment. If red cells are needed urgently before it is resolved, group O red cells are issued.

Anti-A₁ reacts with A₁ cells and spares group O cells and the patient's own cells. Here the O cells and the autocontrol react too.

Coin stacks with reactive O cells and autocontrol point to rouleaux from the myeloma protein. Saline replacement disperses rouleaux and leaves true agglutination in place.

Hint
  1. Look at the group O cells and the autocontrol.
  2. Ask what in myeloma plasma can make cells stick together without antibody.

Review ABO discrepancies

Problem 3 of 3 · On your own

A 58-year-old man's record from last year shows group A. Five days ago he received 4 units of group O red cells at another hospital. Today his cells react 2+ mixed field with anti-A and 0 with anti-B. His plasma reacts 0 with A1 cells and 4+ with B cells. What explains the weak forward reaction?

A mixed field has several causes. His transfusion 5 days ago explains it before any subgroup is considered.

Assigned a subgroup from one weak forward reaction

A weak forward reaction can come from an A subgroup, a mixed red-cell population, immature antigen expression in a neonate, or reagent and method differences. Naming a subgroup from that reaction skips the reverse grouping, lectin testing, and history that separate these causes. In a neonate, a single lectin result does not resolve the subgroup.

Donor O cells stay unagglutinated beside his A cells, which gives a mixed field. His reverse grouping and his record both support group A.

A reagent problem would weaken every reaction evenly. A mixed field shows two cell populations.

The lectin would test a mixture of donor and patient cells. His recent group O transfusion explains the mixed field.

Review A1, A2, and weaker expression

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Evangeline Spinner, 52, a beekeeper, has a type and screen before planned surgery.
  • She has not been transfused or pregnant in the past 3 months, and she has no previous record at this hospital.
  • The antibody screen is negative.
Decision 1 of 3
TestReaction
Anti-A, patient cells4+
Anti-B, patient cells0
A1 cells, patient plasma1+
B cells, patient plasma4+
O cells, patient plasma0
Autocontrol0

What kind of discrepancy is this, and what is the next check?

Only the A₁ cells react unexpectedly, with O cells and the autocontrol nonreactive. The lectin and A₂ cells test for A₂ with anti-A₁.

Group A plasma should not agglutinate A₁ cells. The 1+ reaction must be resolved before a group is recorded.

Assigned ABO from one side of a discrepancy

A weak subgroup, a mixed population after transfusion or transplant, or a missing plasma reaction can each make one side of ABO testing misleading. A group assigned from that side can be wrong and lead to incompatible components, which is why the discrepancy is resolved before assignment. If red cells are needed urgently before it is resolved, group O red cells are issued.

Rouleaux would also make O cells and the autocontrol react. Both are nonreactive.

Review ABO discrepancies

Decision 2 of 3

Surgery moves up before the lectin result is back, and red cells are requested. Which ABO group is issued?

Her group is not recorded until the discrepancy is resolved. A group taken from one side can be wrong.

Assigned ABO from one side of a discrepancy

A weak subgroup, a mixed population after transfusion or transplant, or a missing plasma reaction can each make one side of ABO testing misleading. A group assigned from that side can be wrong and lead to incompatible components, which is why the discrepancy is resolved before assignment. If red cells are needed urgently before it is resolved, group O red cells are issued.

AB red cells carry A antigen and B antigen, and her plasma contains anti-B. They are incompatible.

Red cells needed before an ABO discrepancy is resolved are group O. The investigation continues.

Review ABO discrepancies

Decision 3 of 3

Her cells are nonreactive with anti-A₁ lectin, and her plasma is nonreactive with A₂ cells. What does the pattern show?

An autoantibody would react with her own cells, and the autocontrol is nonreactive.

Her A cells lack A₁, and her plasma reacts with A₁ cells and spares A₂ and O cells. Forward grouping, lectin, selected cells and history agree on A₂ with anti-A₁.

Acquired B shows as an unexpected reaction of the patient's cells with anti-B. Her cells are nonreactive with anti-B, and the extra reaction is in the reverse grouping.

Review A1, A2, and weaker expression

Her plasma reacted only with A1 cells, with group O cells and the autocontrol nonreactive. That pattern suggested anti-A1, and the lectin and A2 cells confirmed an A2 phenotype. Red cells issued before the discrepancy was resolved were group O.

For reference

Sources checked