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Two antibodies, the autocontrol and enzymes

18 min

Skills you'll build2 skills
  • Tell an autoantibody from a new alloantibody by the autocontrol, DAT, and eluate
  • Read an enzyme-treated panel beside the untreated panel

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

Try first

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsIS37AHG
1++00+0++0+++00+002+
2++00+++0++0+++0002+
3+0++00+0+0+0+0+002+
40+0++0+++0+++++002+
500+++0+0++++0++002+
6000++++0++00+0+002+
7000++0++00+++++002+
8+00++0+0+0++0+0002+
9000++0+0+++++0+002+
10+++++0++++0++0+002+
11000++0+++++0+++002+
Autocontrol002+
Try first

A woman has had no transfusion or pregnancy in the past 3 months. Her plasma reacts with every panel cell at the antiglobulin phase, and her autocontrol reacts as strongly. What does this pattern suggest first?

Keep going to see the reasoning.

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.

Get the idea

Use the explanation to check your first decision.

Recognizing more than one antibody

Suspect a second antibody when no single antigen accounts for every reactive cell. Reaction strengths or phases that differ from cell to cell, beyond what dosage explains, also suggest a second antibody.1,2 Work the panel as usual:

  1. Cross out antigens on the nonreactive cells.
  2. Test each antigen that is left against the reactive cells.
  3. If one antigen explains some reactive cells and a second explains the rest, both antibodies are likely present.

Each antibody then needs its own reactive antigen-positive cells and nonreactive antigen-negative cells. The patient should type negative for both antigens.1,2

Interpreting the autocontrol

The autocontrol tests the patient's plasma with the patient's own red cells. It supports the interpretation. The autocontrol result does not prove whether an antibody is an autoantibody or an alloantibody.1

  • Autocontrol nonreactive. An alloantibody is favored.
  • Every cell reactive and the autocontrol reactive, no recent transfusion. A warm autoantibody or a drug effect is favored. A DAT follows, and adsorption is used to detect underlying alloantibodies.1,4
  • Autocontrol reactive, often weak and mixed field, soon after a transfusion. A new alloantibody may be coating surviving donor cells. An eluate from the patient's cells shows the same specificity as the plasma, and the finding can indicate a delayed hemolytic transfusion reaction.1

Enzyme-treated panels

Ficin and papain cut proteins on the red cell surface. Some antigens lose their reactivity and others react more strongly.1,3

Effect of ficin or papainAntigens
EnhancedRh, Kidd, Lewis, P1, I
Destroyed or markedly weakenedDuffy (Fya, Fyb), M, N
VariableS, s
PreservedKell

Read the treated panel beside the untreated one. A reaction that disappears after treatment points to an enzyme-sensitive antigen. A reaction that grows stronger points to an enhanced one.1,3 Treated cells have lost their Duffy, M and N antigens. Exclusions for those antibodies therefore come from untreated cells.1,3

Follow the bench demonstration

Sources for this step
References
  1. Bloch EM, Campbell-Lee S, McKenna DH Jr, Montemayor-Garcia C, Schwartz J, Shaz B, Storry J, eds. Technical Manual. 22nd ed. AABB; 2026.
  2. 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
  3. Raman L, Armstrong B, Smart E. Principles of laboratory techniques. ISBT Sci Ser. 2020;15(suppl 1):81-111. doi:10.1111/voxs.12591
  4. Johnson ST, Puca KE. Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies. Hematology Am Soc Hematol Educ Program. 2022;2022(1):96-104. doi:10.1182/hematology.2022000406

Watch one

Read each decision and its explanation.

A man transfused 2 years ago has a reactive antibody screen. His plasma is tested with an 11-cell panel by tube antiglobulin test. His autocontrol is nonreactive.

The same cells treated with ficin give these antiglobulin reactions: cells 3, 5 and 10 react 3+ to 4+. Every other cell is nonreactive. Which antibodies are present?

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsIS37AHG
1++00+0++0+++00+002+
2++00+++0++0+++0000
3+0++00+0+0+0+0+0w+3+
40+0++0+++0+++++001+
500+++0+0++++0++002+
6000++++0++00+0+000
7000++0++00+++++002+
8+00++0+0+0++0+0000
9000++0+0+++++0+000
10+++++0++++0++0+0w+3+
11000++0+++++0+++001+
Autocontrol000
  1. Read the autocontrol first. It is nonreactive at every phase.

    A nonreactive autocontrol favors alloantibodies over an antibody that also coats his own cells.

  2. Cross out on the nonreactive cells 2, 6, 8 and 9. D, C, c, e, K, k, Fyb, Jka, Jkb, M, N, S and s are crossed out. E and Fya remain.

    A dosage antigen is crossed out only on a nonreactive cell homozygous for it, such as the Jk(a+b−) cells 2 and 6 for Jka.

  3. Test E against the reactive cells. It explains cells 3, 5 and 10. Cells 1, 4, 7 and 11 react and lack E.

    An antigen that leaves reactive cells unexplained means a second antibody is present.

  4. Test Fya. It explains cells 1, 4, 7 and 11. The Fy(a+b−) cells 1 and 7 react 2+, and the Fy(a+b+) cells 4 and 11 react 1+.

    Stronger reactions on homozygous cells are the dosage pattern of a Duffy antibody.

  5. Read the ficin panel beside the untreated one. The E-positive cells react more strongly. The cells carrying Fya without E become nonreactive.

    Ficin enhances Rh antigens and destroys Duffy antigens, so the treated panel separates the two antibodies.

  6. Keep anti-Fya in the answer. The ficin panel cannot show anti-Fya because the antigen has been destroyed.

    Treated cells have lost Fya, so their lack of reaction cannot exclude anti-Fya.

  7. Confirm. E has 3 reactive positive cells and 4 nonreactive negative cells. Fya has 5 and 4. His red cells should type E-negative and Fy(a−).

    Each antibody needs its own reactive and nonreactive cells, and he cannot make an antibody to an antigen he carries.

Anti-E and anti-Fya. The ficin panel separated them. The E reactions were enhanced, and the Fya reactions disappeared because ficin destroyed Fya.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A patient's plasma is nonreactive with every cell of a papain-treated panel. Which antibody could still be present?

Incorrect. Papain enhances Kidd antigens, so a Kidd antibody such as anti-Jka would react more strongly with the treated cells.

Incorrect. Papain enhances Rh antigens, so an Rh antibody would react more strongly with the treated cells.

Correct. Papain and ficin destroy Duffy antigens, so treated cells cannot react with anti-Fya or anti-Fyb. Untreated antigen-positive cells are needed to exclude a Duffy antibody.

Hint
  1. Sort the blood group systems by what papain does to them: enhanced, destroyed or preserved.
  2. A treated cell that has lost an antigen cannot react with its antibody, whether the antibody is present or not.

Review Reagent effects and antibody patterns

Problem 2 of 3 · Hints available

A woman's plasma reacts with 5 of 11 panel cells at the antiglobulin phase. Her autocontrol is nonreactive. With ficin-treated cells, cells 3, 5 and 9 react more strongly, and cells 2 and 6 react as before. Cross out what the nonreactive cells rule out, then name the antibodies.

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsIS37AHG
1++00+0++0+0+0+0000
2++00+++0+0+++0+002+
3+0++00+++++0+++0w+3+
40+0++0+0+0+++0+000
500+++0++0+++0++002+
6000+++++++00+++002+
7000++0+0+++++0+000
8+00++0+++0+0+++000
9+0+++++0++0++0+003+
10000++0++00++00+000
11000++0+0++00++0000
Autocontrol000
Hint
  1. Start with the nonreactive cells. Cross out C, c, E, e, Duffy, Kidd or MNS antigens only on a cell homozygous for them. Cross out D and K on any nonreactive cell.
  2. Two antigens are left. Check which reactive cells each one explains.
  3. Compare what ficin did to cells 2 and 6 with what it did to cells 3 and 5.
Show the answer and the reasoning

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

Cell 4 is nonreactive and rules out e, k, Fyb, Jkb, s.

Cell 7 is nonreactive and rules out c, e, k, Fyb, s.

Cell 8 is nonreactive and rules out D, c, e, k, Jkb, N.

Cell 10 is nonreactive and rules out c, e, k, Fya, Jkb, M, s.

Cell 11 is nonreactive and rules out c, e, k, Fyb, Jka, N, S.

Anti-E, Anti-K

Anti-E and anti-K. Cells 3, 5 and 9 carry E, and cells 2, 6 and 9 carry K. The six nonreactive cells rule out every other antigen on the panel. Ficin strengthened the E reactions because it enhances Rh antigens, and it left the K reactions unchanged because ficin does not affect Kell antigens.

Review Enzymes, enhancement media, and lectins

Problem 3 of 3 · On your own

A man's plasma reacts with 7 of 11 panel cells at the antiglobulin phase. His autocontrol is nonreactive. With ficin-treated cells, cells 2, 5, 8, 10 and 11 react 2+ to 3+, and cells 4 and 7 are nonreactive. Name the antibodies.

Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsIS37AHG
1++00+0+0++0+00+000
2++00++++00+++++003+
3+0++00+0++00++0000
40+0++0++++0++0+001+
500+++0+0++++0++001+
6000++++0++00+++000
7000++0++0+0++0+002+
8+00++0+0+0++0++002+
9000++0+0++0++0+000
10++0++0+++0+0++0003+
11000++0+0+++++++001+
Autocontrol000
Show the answer and the reasoning

Cell 1 is nonreactive and rules out D, C, e, k, Fyb, Jka, M, s.

Cell 3 is nonreactive and rules out D, E, c, k, Fyb, Jka, N, S.

Cell 6 is nonreactive and rules out c, e, K, k, Fyb, Jka, N.

Cell 9 is nonreactive and rules out c, e, k, Fyb, Jka, s.

Anti-Fya, Anti-Jkb

Anti-Fya and anti-Jkb. The nonreactive cells 1, 3, 6 and 9 rule out every other antigen. Cells 4 and 7 carry Fya without Jkb, and ficin removed their reactions because it destroys Duffy antigens. That loss fits anti-Fya and cannot exclude it. The Jkb-positive cells kept reacting after ficin because it enhances Kidd antigens, and the Jk(a−b+) cells 2, 8 and 10 react more strongly than the Jk(a+b+) cells 5 and 11.

Review Enzymes, enhancement media, and lectins

Use it

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

  • Perry Sites, 71, a retired letter carrier (MRN 5520931), received 2 units of red cells 8 days ago after hip surgery.
  • His antibody screen before that transfusion was nonreactive.
  • Today his hemoglobin is 7.9 g/dL, down from 10.1 g/dL two days after the transfusion. A new specimen arrives with an order for 2 more units.
  • Today's antibody screen reacts at the antiglobulin phase.
  • His autocontrol reacts weakly and looks mixed field, with small agglutinates among many free cells.
Antibody panel: + the cell carries the antigen, 0 it lacks it
CellDCEceKkFyaFybJkaJkbMNSsIS37AHG
1++00+0++0+++00+001+
2++00+++0++0+++0002+
3+0++00+0+0+0+0+000
40+0++0+++0+++++000
500+++0+0++++0++001+
6000++++0++00+0+002+
7000+++++00+++++000
8++00+0+0+0++0+0000
9000++0+0+++++0+001+
10+++++0++++0++0+002+
11000++0+++++0+++001+
Autocontrol00w+
Decision 1 of 3

Here is today's panel. What does the weak mixed-field autocontrol most likely show?

A warm autoantibody usually reacts with every panel cell and gives an even autocontrol reaction. Here only the Jk(a+) cells react, and the autocontrol is mixed field 8 days after a transfusion.

Called a reactive autocontrol autoimmune after transfusion

After a recent transfusion, a new alloantibody can coat surviving donor cells and make the autocontrol and DAT reactive, sometimes mixed field. Calling that an autoantibody misses the alloantibody and a possible delayed hemolytic reaction. The transfusion history, the reaction pattern, and an eluate separate the two.

The reactions are at the antiglobulin phase only, with nothing at immediate spin. That is the pattern of an IgG antibody.

Every reactive cell carries Jka, and the Jk(a+b−) cells react more strongly. Only the donor cells that carry Jka are coated, so the autocontrol shows a mixed field.

Review Pattern-based problem solving

Decision 2 of 3

Which testing shows what is coating his red cells?

Adsorption removes antibody from plasma so the plasma can be tested further. It does not show what coats the cells, and his specimen contains donor cells that would adsorb the alloantibody.

The DAT shows what kind of protein coats the cells. The eluate recovers that antibody so its specificity can be compared with the plasma's.

Ficin enhances Kidd antigens and would strengthen the plasma reactions. It retests the plasma and gives no information about his cells.

His specimen still contains donor cells, so a serologic type would test the mixture. His Jka status comes from a pretransfusion specimen or genotyping.

Review Pattern-based problem solving

Decision 3 of 3

The DAT is IgG-positive, weak and mixed field. The eluate reacts only with the Jk(a+) cells, and the last wash is nonreactive. What does the laboratory do?

The same antibody is free in the plasma and bound to donor cells, with a falling hemoglobin. The physician is notified under the transfusion reaction procedure, and his 2 new units are Jk(a−) with an antiglobulin crossmatch.

The eluate has a single specificity, and his pretransfusion screen was nonreactive. Calling it an autoantibody would miss the anti-Jka and a possible delayed hemolytic transfusion reaction.

Called a reactive autocontrol autoimmune after transfusion

After a recent transfusion, a new alloantibody can coat surviving donor cells and make the autocontrol and DAT reactive, sometimes mixed field. Calling that an autoantibody misses the alloantibody and a possible delayed hemolytic reaction. The transfusion history, the reaction pattern, and an eluate separate the two.

The eluate result shows the antibody is on the transfused cells. That finding belongs in the report and the reaction investigation.

Review Pattern-based problem solving

A mixed-field autocontrol 8 days after a transfusion showed that only part of his red cells were coated, the donor cells carrying Jka. The eluate showed the same anti-Jka as the plasma panel.

For reference

Sources checked