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Reaction strength, complement and the antiglobulin test

16 min

Skills you'll build4 skills
  • Predict how method, enhancement medium, and cell concentration change a reaction grade
  • Tell complement coating from proven hemolysis on a C3d-positive DAT
  • Tell in vivo coating (DAT) from in vitro coating (IAT) and what each reagent detects
  • Reject a negative tube antiglobulin test when required control cells fail

At the bench: Use the antibody workup to select suitable donor units · Perform a direct antiglobulin workup and interpret its controls

Try first

Try first

A patient's EDTA red cells are washed and tested with anti-IgG. They agglutinate 2+. What does this result show?

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.

Agglutination and enhancement

Red cells carry a negative surface charge that keeps them apart. IgM is a large pentamer that can span that gap and agglutinate cells directly. IgG is smaller and usually coats the cells without clumping them.1 Low-ionic-strength solution (LISS) speeds antibody uptake. Polyethylene glycol (PEG) crowds antibody around the cells and detects weak IgG.1

A reaction grade is specific to its method. The enhancement medium, cell concentration and endpoint all change the grade, so a 2+ in a tube and a 2+ in a column are different measurements. Compare grades within one method.1,2

The direct and indirect tests

Antihuman globulin (AHG) binds the human globulins named on its label. Polyspecific reagent detects IgG and C3d. Monospecific anti-IgG and anti-C3d show which one coats the cells.3

TestWhat is testedWhere the coating happened
Direct antiglobulin test (DAT)The patient's washed red cells, from EDTA bloodIn the patient's circulation
Indirect antiglobulin test (IAT)The patient's plasma with reagent or donor red cells, incubated at 37 °CIn the tube

A positive DAT shows globulin on the red cells. It does not show red cell destruction or identify the antibody.1

Complement coating

Antigen-bound IgM, and IgG clustered closely enough, activate the classical complement pathway and leave C3 fragments on the cell. C3d can remain on the cell after the antibody has dissociated, so a DAT can react with anti-C3d alone.1,4 EDTA blood is used because EDTA stops complement from attaching after collection.1 Red cell destruction is shown by other results: a falling hemoglobin, raised indirect bilirubin and lactate dehydrogenase (LDH), and a low haptoglobin.1

Check cells

A negative tube antiglobulin test is valid only when IgG-coated check cells, added afterward, agglutinate. When they do not agglutinate, unbound globulin left by poor washing may have neutralized the AHG. The negative result is invalid, and the test is repeated.3,5

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. Raman L, Armstrong B, Smart E. Principles of laboratory techniques. ISBT Sci Ser. 2020;15(suppl 1):81-111. doi:10.1111/voxs.12591
  3. Diagast. Anti-Human Globulin Anti-IgG, Anti-C3d, and Anti-IgG,-C3d Instructions for Use. US Food and Drug Administration. Accessed September 27, 2026.
  4. Arthur CM, Chonat S, Fasano R, et al. Examining the role of complement in predicting, preventing, and treating hemolytic transfusion reactions. Transfus Med Rev. 2019;33(4):217-224. doi:10.1016/j.tmrv.2019.09.006
  5. Alba Bioscience Limited. Anti-Human Globulin Anti-IgG Instructions for Use. US Food and Drug Administration. Accessed September 27, 2026.

Watch one

Read each decision and its explanation.

A patient's plasma is tested by tube indirect antiglobulin test against screening cell I, which is Fy(a+b−). The plasma contains IgG anti-Fya. Follow the tube through the test. What does it read, and what would make a negative tube valid?

  1. Incubate the plasma with the reagent cells at 37 °C. The IgG anti-Fya binds Fya on the cells, and the tube shows no clumping.

    IgG is too small to bridge two red cells, so binding shows no visible clumping.

  2. Wash the cells three times with saline.

    Unbound globulin left in the tube would neutralize the antihuman globulin reagent.

  3. Add antihuman globulin and centrifuge. The cells agglutinate 2+, a positive result.

    The reagent binds the IgG on neighboring cells and bridges them into a lattice.

  4. For a negative tube only, add IgG-coated check cells. They must agglutinate.

    Agglutination of the check cells shows the AHG was active and the washing was adequate.

The tube reads 2+ at the antiglobulin phase. The IgG bound to the cells in the tube, which makes this an indirect test. A positive tube needs no check cells. A negative tube is valid only when the check cells agglutinate.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

An EDTA specimen's direct antiglobulin test (DAT) reacts with anti-C3d and not with anti-IgG. Which explanation fits this pattern?

Incorrect. EDTA chelates the calcium and magnesium that complement activation needs, so it limits activation after collection. The C3d was bound in vivo.

Incorrect. Some Kidd antibodies bind complement, but a DAT shows only what is on the cells. Identifying the antibody takes eluate and plasma antibody studies.

Correct. An antibody such as IgM can activate complement on the cell and then dissociate, leaving C3d bound to react with anti-C3d. The pattern shows complement coating. The history, hemolysis markers, and antibody results show whether hemolysis accompanies it.

Hint
  1. EDTA binds calcium and magnesium. Ask what complement needs to activate.
  2. A DAT shows only what is on the cells at collection. Ask what each option claims beyond that.

Review Complement activation and hemolysis

Problem 2 of 3 · Hints available

A conventional tube indirect antiglobulin test (IAT) is nonreactive, and the required IgG-sensitized control cells added afterward also fail to agglutinate. What follows?

Incorrect. A negative tube IAT is valid only when the IgG-sensitized control cells agglutinate. They did not, so the patient tube may be negative because the antihuman globulin was neutralized.

Correct. Nonreactive check cells show that no active antihuman globulin (AHG) remained, often because inadequate washing left unbound globulin that neutralized it. The negative result is invalid, so the test is repeated from the required step with the complete wash sequence.

Incorrect. A reaction with extra check cells shows only that some AHG activity remained after they were added. It cannot show that active AHG was present when the patient tube was read, so the failed test is repeated.

Hint
  1. Ask what the check cells show when they agglutinate.
  2. Ask when active reagent had to be present for the patient tube to be valid.

Review Tube-test controls and failure points

Problem 3 of 3 · On your own

Last month a patient's anti-E reacted 2+ with E-positive cells in a LISS tube test. Today another laboratory tests a new specimen by column agglutination and reports 3+. What does the change in grade show?

The two grades come from different methods. Cell concentration, medium and endpoint differ, so the grades cannot show a change in the antibody.

Compared reaction grades across different methods

Reaction grade depends on the method, enhancement medium, cell concentration, and endpoint, so a 2+ tube reaction and a 2+ column reaction measure different things. Reading a change between methods as a change in the antibody can misjudge a trend or a workup.

Each validated method reports on its own scale. The same antibody can grade higher in a more sensitive method.

A grade is specific to its method. A trend needs the same method, medium and endpoint each time.

Review Reaction strength

Use it

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

  • Walter Frost, 71, a retired letter carrier who still walks his old route, MRN 6052913, has a DAT and a type and screen.
  • EDTA DAT: polyspecific 2+, anti-IgG 0, anti-C3d 2+.
  • Tube antibody screen: both cells 0 at the antiglobulin phase. The check cells added afterward are 0.
  • His haptoglobin, LDH and indirect bilirubin on the same draw are within their reference intervals.
Decision 1 of 3

What happens to the antibody screen?

The negative reading is valid only when the check cells agglutinate. They did not, so the AHG may have been neutralized.

Reported a negative tube test despite failed check cells

IgG-sensitized check cells show that active antihuman globulin remained in a negative tube. When they fail to agglutinate, unbound globulin left by inadequate washing may have neutralized the reagent, so the negative patient result could be false and the test is repeated.

Extra check cells show only that some AHG remains now. They cannot show it was active when the patient tubes were read.

A comment does not make an invalid test valid. The screen could be falsely negative.

Failed check cells make the negative screen invalid. The repeat uses the full washing so no unbound globulin is left to neutralize the AHG.

Review Tube-test controls and failure points

Decision 2 of 3

How is his DAT interpreted?

Only anti-C3d reacted, so complement coats his cells. His hemolysis markers show no red cell destruction.

The DAT shows complement on the cells. His haptoglobin, LDH and indirect bilirubin are within their reference intervals and give no evidence of destruction.

Reported C3d coating as intravascular hemolysis

A C3d-only DAT shows complement on the red cells, often left behind after an antibody dissociated. Destruction is shown separately by a falling hemoglobin, increased indirect bilirubin and lactate dehydrogenase, and decreased haptoglobin, so reporting the DAT as hemolysis states more than the test measured.

Anti-IgG is nonreactive. Only C3d is detected.

Review Complement activation and hemolysis

Decision 3 of 3

The repeated screen is 0 with both cells, and the check cells agglutinate 2+. What do the DAT and the screen show together?

The screen tests his plasma against reagent cells. His own cells carry C3d, as the DAT showed.

The DAT detects coating that occurred in the circulation. The valid indirect test detected no antibody to the antigens on the screening cells.

The screen and the DAT use different red cells. The DAT tested his own cells, washed straight from the EDTA tube.

Review Antihuman globulin reagents

Each result in this case is interpreted by what its reagent detects. The check cells showed whether the AHG was active when the screen was read. Anti-C3d showed complement on his cells, and his hemolysis markers within their reference intervals showed no red cell destruction.

For reference

Sources checked