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Identifying one antibody

18 min

Skills you'll build1 skill
  • Use antigen-positive nonreactive cells for valid antibody exclusions

At the bench: Use the antibody workup to select suitable donor units

Try first

Try first

Two panel cells are nonreactive. Cell 4 is Jk(a+b+) and cell 7 is Jk(a−b+). Which of them lets you cross out anti-Jkb?

Keep going to see the reasoning.

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.

Exclusion and identification

An antibody panel tests the patient's plasma against 8 to 16 group O cells, each with its antigens listed on the lot's antigram. Work the panel in this order:1,2

  1. Take a nonreactive cell. The plasma contains no detectable antibody against the antigens that cell carries.
  2. Cross out each antigen that cell carries in a form the antibody would detect.
  3. Repeat for every nonreactive cell.
  4. Compare the antigens not crossed out with the reactive cells. The antigen whose pattern matches the reactions identifies the antibody.

Double-dose cells

Antibodies to C, c, E, e, Jka, Jkb, Fya, Fyb, M, N, S and s often show dosage. They react more strongly with cells that carry a double dose of the antigen, and a weak example can miss a single dose.3,4 Under the usual exclusion rule, cross out one of these antigens only on a nonreactive cell that carries it without its partner, such as Jk(a+b−) for Jka. D, K, k, P1, Lea and Leb are crossed out on any nonreactive cell that carries them. Your laboratory's validated exclusion rule sets the details.2

An antigen that is not on any suitable nonreactive cell cannot be excluded. Selected cells from another lot, or the patient's own antigen type, resolve it.2

Confirming the answer

A presumptive specificity needs two checks.2,5

  • The rule of three. At least three reactive cells carry the antigen, and at least three nonreactive cells lack it.
  • The patient's phenotype. The patient's red cells lack the antigen. Recent transfusion or a positive DAT can make this typing unreliable, and genotyping may be needed.

The patient then receives red cells negative for the antigen, crossmatched through the antiglobulin phase.2

Follow the bench demonstration

Sources for this step
References
  1. Testing of source material, 21 CFR §660.33 (2026). Accessed September 27, 2026. https://www.ecfr.gov/current/title-21/chapter-I/subchapter-F/part-660/subpart-D/section-660.33
  2. Bloch EM, Campbell-Lee S, McKenna DH Jr, Montemayor-Garcia C, Schwartz J, Shaz B, Storry J, eds. Technical Manual. 22nd ed. AABB; 2026.
  3. Immucor, Inc. Panoscreen Reagent Red Blood Cells Package Insert. US Food and Drug Administration. Accessed September 27, 2026.
  4. Raman L, Armstrong B, Smart E. Principles of laboratory techniques. ISBT Sci Ser. 2020;15(suppl 1):81-111. doi:10.1111/voxs.12591
  5. Manduzio P. Alloantibody identification: the importance of temperature, strength reaction and enzymes—a practical approach. Hematol Rep. 2024;16(4):815-824. doi:10.3390/hematolrep16040077

Watch one

Read each decision and its explanation.

Marguerite Frost, 45, has a reactive antibody screen before a hysterectomy. Her panel reads:

Which antibody is present?

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsP1LeaLebIS37AHG
1++00+++++++++0++0+001+
2++00+0+0++00++00+0000
3+0++00++00++00++0+002+
4+00++0+00+0++0++00000
50+0++0++00++0++00+002+
600+++0+0+++0+0++0+000
7000+++++0+0+0+0++0002+
8000++0+0+0++++++0+000
9000++++0+++0+0+00+000
10++0++0++00++++++0+002+
11+0++00+0++0+00+++0000
Autocontrol000
  1. Check the autocontrol. It is 0 in every phase.

    A nonreactive autocontrol favors an alloantibody over an autoantibody.

  2. Note the phase. Every reaction is at the antiglobulin phase.

    IgG antibodies such as Rh, Kell, Kidd and Duffy react at the antiglobulin phase.

  3. Cross out on the nonreactive cells 2, 4, 6, 8, 9 and 11. Every antigen except Fya is crossed out on one of them.

    A nonreactive cell shows no detectable antibody against what it carries.

  4. Check the dosage antigens. E is crossed out only on cell 11, which is E+e−, and Jkb only on cell 8, which is Jk(a−b+). Both are double-dose cells, so both exclusions are valid.

    A single-dose cell could miss a weak antibody that shows dosage.

  5. Match the pattern. Fya is on cells 1, 3, 5, 7 and 10, the five reactive cells. Cell 1 is Fy(a+b+) and reacts 1+, and the double-dose cells react 2+, which fits a Duffy antibody.

    Every reactive cell must carry the antibody's antigen.

  6. Confirm. Five reactive Fy(a+) cells and six nonreactive Fy(a−) cells meet the rule of three. Type her red cells for Fya.

    An alloantibody forms only against an antigen the patient lacks.

The panel identifies anti-Fya. When her red cells type Fy(a−), she receives Fy(a−) red cells crossmatched through the antiglobulin phase.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A patient's screen reacted at the antiglobulin phase. Cross out what the nonreactive cells rule out, then name the antibody.

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsP1LeaLebIS37AHG
1++00+0++0+0+0+0+0+000
2++00+++0+0+0+0+0+0000
3+0++00++00++++++0+003+
40+0++0+0++0+00++0+000
500+++0+++++0++000+002+
6000++++0+++++0+++0000
7000++0++00++0+0+0+000
8000++0+0++0++0+000000
9+00++0++++0+0++++0000
10++0++0++00+0+0++0+000
11+0+++0+0++++00+00+002+
Autocontrol000
Hint
  1. Start with a nonreactive cell that carries many antigens, such as cell 1. Cross out only what it carries in a double dose, plus D, K, k, P1 and Lewis.
  2. Look at the strongest reaction, and read that cell's zygosity for each antigen not yet crossed out.
Show the answer and the reasoning

Cell 1 is nonreactive and rules out D, C, e, k, Fya, Jka, M, S, P1, Leb.

Cell 2 is nonreactive and rules out D, C, e, K, k, Fyb, Jkb, N, s, Lea.

Cell 4 is nonreactive and rules out e, k, Fyb, Jka, M, s, P1, Leb.

Cell 6 is nonreactive and rules out c, e, K, k, Fyb, s, P1, Lea.

Cell 7 is nonreactive and rules out c, e, k, Fya, Jkb, M, S, P1, Leb.

Cell 8 is nonreactive and rules out c, e, k, Fyb, Jka, s.

Cell 9 is nonreactive and rules out D, c, e, k, Jka, M, P1, Lea.

Cell 10 is nonreactive and rules out D, e, k, Fya, Jkb, N, s, P1, Leb.

Anti-E

Every antigen except E is crossed out on a suitable nonreactive cell. Cells 3, 5 and 11 carry E and react, and cell 3, which is E+e−, reacts strongest. Eight nonreactive cells lack E, so the pattern meets the rule of three for anti-E.

Review Antibody investigation

Problem 2 of 3 · Hints available

A patient transfused 2 weeks ago has a new reactive screen. Cross out what the nonreactive cells rule out, then name the antibody.

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsP1LeaLebIS37AHG
1++00+0++0+0+0+0+0+002+
2++00+++0+0+0+0+0+0000
3+0++00++00++++++0+000
40+0++0+0++0+00++0+002+
500+++0+++++0++000+00w+
6000++++0+++++0+++0000
7000++0++00++0+0+0+000
8000++0+0++0++0+000002+
9+00++0++++0+0++++0002+
10++0++0++00+0+0++0+000
11+0+++0+0++++00+00+00w+
Autocontrol000
Hint
  1. Before you cross out an antigen, check that the nonreactive cell carries it without its partner.
  2. Compare the grade of each reactive cell with its zygosity for the antigens not crossed out.
Show the answer and the reasoning

Cell 2 is nonreactive and rules out D, C, e, K, k, Fyb, Jkb, N, s, Lea.

Cell 3 is nonreactive and rules out D, E, c, k, Fya, Jkb, P1, Leb.

Cell 6 is nonreactive and rules out c, e, K, k, Fyb, s, P1, Lea.

Cell 7 is nonreactive and rules out c, e, k, Fya, Jkb, M, S, P1, Leb.

Cell 10 is nonreactive and rules out D, e, k, Fya, Jkb, N, s, P1, Leb.

Anti-Jka

Jka is on every reactive cell. The Jk(a+b−) cells 1, 4, 8 and 9 react 2+, and the Jk(a+b+) cells 5 and 11 react only w+. Cell 6 is Jk(a+b+) and nonreactive. A weak anti-Jka can miss a single dose, so Jka cannot be crossed out on cell 6.

Review Antibody investigation

Problem 3 of 3 · On your own

A patient with a reactive screen is scheduled for surgery. Work the panel and name the antibody.

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsP1LeaLebIS37AHG
1++00+++++++++0++0+003+
2++00+0+0++00+++0+0000
3+0++00++00++00++0+000
4+00++0+00+0++0++00000
50+0++0++00++0++00+000
600+++0+0+++0+0++0+000
7000+++++0+0+0+0++0003+
8000++0+0+0++++++0+000
9000++++0+++0+0+00+003+
10++0++0++00++++++0+000
11+0++00+0++0+00+++0000
Autocontrol000
Show the answer and the reasoning

Cell 2 is nonreactive and rules out D, C, e, k, Fyb, Jka, N, Lea.

Cell 3 is nonreactive and rules out D, E, c, k, Fya, Jkb, M, s, P1, Leb.

Cell 4 is nonreactive and rules out D, c, e, k, Jka, s, P1.

Cell 5 is nonreactive and rules out e, k, Fya, Jkb, M, Leb.

Cell 6 is nonreactive and rules out c, k, Fyb, N, s, P1, Leb.

Cell 8 is nonreactive and rules out c, e, k, Fyb, Jkb, P1, Leb.

Cell 10 is nonreactive and rules out D, e, k, Fya, Jkb, P1, Leb.

Cell 11 is nonreactive and rules out D, E, c, k, Fyb, Jka, M, s, P1, Lea.

Anti-K

Cells 1, 7 and 9 carry K and react 3+, and eight nonreactive cells lack K, so the pattern fits anti-K. S cannot be excluded, because every nonreactive S+ cell also carries s. Selected S+s− cells or the patient's S type will resolve it.

Review Antibody investigation

Use it

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

  • Esperanza Maxwell, 52, a choir director who hums through her blood draws, MRN 3319826, has a reactive antibody screen.
  • Her knee replacement is in 3 days.
  • She has had two pregnancies and no transfusions.
  • Her panel's autocontrol is nonreactive.
Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsP1LeaLebIS37AHG
1++00+++++++++0++0+000
2++00+0+0++00++00+0002+
3+0++00++00++00++0+000
4+00++0+00+0++0++00000
50+0++0++00++0++00+001+
600+++0+0+++0+0++0+000
7000+++++0+0+0+0++0002+
8000++0+0+0++++++0+001+
9000++++0+++0+0+00+000
10++0++0++00++++++0+001+
11+0++00+0++0+00+++0000
Autocontrol000
Decision 1 of 3

Which antibody does her panel identify?

Cell 3 is Fy(a+b−) and nonreactive, so Fya is crossed out. Cell 2 reacts and lacks Fya.

Cell 3 is M+N− and nonreactive, so M is crossed out. Cell 2 reacts and lacks M.

Every reactive cell carries S, and every other antigen is crossed out on a suitable nonreactive cell. The S+s− cells 2 and 7 react 2+, and the S+s+ cells react 1+.

Cell 2 reacts and is Jk(a+b−), so its reaction cannot come from anti-Jkb. Cell 3, Jk(a−b+), is nonreactive.

Review Antibody investigation

Decision 2 of 3

The pattern meets the rule of three. Which result completes the identification?

An alloantibody forms against an antigen she lacks. She has not been transfused, so her own antigen typing is reliable.

Enzymes weaken or destroy S to a variable degree. Treated cells add little to an anti-S that the untreated panel already shows.

The same cells give the same pattern. Her own antigen type is the confirmation still needed.

Review Antibody investigation

Decision 3 of 3

Her red cells type S−s+. Which red cells are prepared for surgery?

A clinically significant antibody excludes her from the electronic crossmatch. She needs antigen-negative units.

Anti-S is usually IgG and can cause hemolytic transfusion reactions. S-negative units with an antiglobulin crossmatch protect her.

She carries s and cannot form anti-s. Her antibody targets S.

Review Unit selection and availability

Every reactive cell carried S, and every other antigen was crossed out on a suitable nonreactive cell. Her S-negative type confirmed anti-S, so she receives S-negative units crossmatched through the antiglobulin phase.

For reference

Sources checked