Adsorption, elution, thiols and mixed fields

18 min

Skills you'll build4 skills

Choose autologous or allogeneic adsorption from the recent transfusion history
Interpret an eluate against its last-wash control
Read DTT-treated test results knowing which antigens DTT destroys
Choose molecular follow-up when serologic phenotyping is unreliable

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

Try first

A man's warm autoantibody reacts with every panel cell. He was last transfused 2 years ago, and his hematocrit gives enough red cells for adsorption. Which adsorption is the first choice to detect underlying alloantibodies?

Allogeneic adsorption with selected donor cells

Keep going to see the reasoning.

Autologous adsorption with his own red cells

Right. Keep going to follow the reasoning.

Rabbit erythrocyte stroma adsorption

Keep going to see the reasoning.

No adsorption, because every unit will be incompatible anyway

Keep going to see the reasoning.

I don't know yet

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Autoantibody adsorption

A warm autoantibody that reacts with every cell can mask an alloantibody. Adsorption removes the autoantibody from the plasma, and the adsorbed plasma is tested again.^1,2

Autologous adsorption uses the patient's own red cells. It needs a patient with no transfusion in the past 3 months and enough red cells.
Allogeneic adsorption uses selected donor cells of known phenotypes. It is used after a transfusion in the past 3 months, because donor cells still circulating in the patient would remove an alloantibody along with the autoantibody. The cells are selected so that each common clinically significant alloantibody remains in at least one adsorbed aliquot.^1,2

Elution and the last wash

Elution frees antibody bound to red cells. The recovered fluid, the eluate, is tested with panel cells like a plasma.^1,3 The cells are washed before elution, and the last wash is tested beside the eluate. A reactive last wash means free plasma antibody was carried over, and the eluate cannot be interpreted.³ With a nonreactive last wash, a specific eluate after a transfusion identifies an alloantibody bound to donor cells. An eluate reacting with every cell fits a warm autoantibody.^1,2

DTT and anti-CD38 drugs

Daratumumab and other anti-CD38 drugs bind CD38 on reagent red cells, so the plasma reacts with every cell at the antiglobulin phase. Treating the reagent cells with dithiothreitol (DTT) destroys CD38 and removes the interference.⁴ DTT also destroys Kell antigens, so a DTT-treated screen cannot detect anti-K. K-negative units are given unless the patient is known to be K-positive. Antibodies to k, Yt^a and Dombrock antigens can also be missed.⁴

Typing a mixed population

After a recent transfusion, the specimen contains donor and patient red cells. A serologic phenotype tests the mixture and can show antigens the patient lacks. A strongly positive DAT can also distort typing.¹ Red cell genotyping predicts the patient's antigens from DNA and avoids both problems. Reference laboratories can also type a separated fraction of the patient's own young red cells.^1,5

Follow the bench demonstration

Sources for this step

References

Bloch EM, Campbell-Lee S, McKenna DH Jr, Montemayor-Garcia C, Schwartz J, Shaz B, Storry J, eds. Technical Manual. 22nd ed. AABB; 2026.
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
Raman L, Armstrong B, Smart E. Principles of laboratory techniques. ISBT Sci Ser. 2020;15(suppl 1):81-111. doi:10.1111/voxs.12591
Association for the Advancement of Blood & Biotherapies. Association Bulletin #16-02: Mitigating the Anti-CD38 Interference With Serologic Testing. Revised March 2026. Accessed September 27, 2026. https://www.aabb.org/docs/default-source/default-document-library/resources/association-bulletins/ab16-02-revised.pdf?sfvrsn=bd0f61d2_14
Association for the Advancement of Blood & Biotherapies. Standards for Molecular Testing for Red Cell, Platelet, and Neutrophil Antigens. 7th ed. AABB; 2024.

Watch one

Read each decision and its explanation.

A woman transfused 10 days ago has a direct antiglobulin test (DAT) that is IgG-positive, 1+ and mixed field. An acid eluate from her red cells and the last wash are tested with the panel at the antiglobulin phase. The last wash is nonreactive with every cell. The eluate:

What does the eluate show?

Antibody panel: + the cell carries the antigen, 0 it lacks it
Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	+	+	0	0	+	2+
2	+	+	0	0	+	+	+	0	+	+	0	+	+	+	0	0
3	+	0	+	+	0	0	+	0	+	0	+	0	+	0	+	0
4	0	+	0	+	+	0	+	+	+	0	+	+	+	+	+	1+
5	0	0	+	+	+	0	+	0	+	+	+	+	0	+	+	0
6	0	0	0	+	+	+	+	0	+	+	0	0	+	0	+	0
7	0	0	0	+	+	0	+	+	0	0	+	+	+	+	+	2+
8	+	0	0	+	+	0	+	0	+	0	+	+	0	+	0	0
9	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
10	+	+	+	+	+	0	+	+	+	+	0	+	+	0	+	1+
11	0	0	0	+	+	0	+	+	+	+	+	0	+	+	+	1+

Read the last wash first. It is nonreactive with every cell, so the eluate can be interpreted.
A reactive last wash would mean carried-over plasma antibody and would invalidate the eluate.
Look at the reaction pattern. The eluate reacts with 5 of 11 cells.
An eluate reacting with every cell fits an autoantibody. Reactions with only some cells suggest a specificity.
Cross out on the nonreactive cells 2, 3, 5, 6, 8 and 9. Every antigen except Fy^a is excluded.
The eluate is read with the same exclusion rules as a plasma.
Check the reactive cells. All 5 carry Fy^a. The Fy(a+b−) cells 1 and 7 react 2+, and the Fy(a+b+) cells react 1+.
Stronger reactions on homozygous cells fit a Duffy antibody.
Confirm her type from a pretransfusion specimen or by genotyping. She is Fy(a−).
She cannot make anti-Fy^a if she carries Fy^a, and a posttransfusion specimen contains donor cells.

The eluate contains anti-Fy^a that was bound to her transfused Fy(a+) donor cells. The nonreactive last wash shows the eluate is valid. It is reported, and the transfusion service physician is notified of a possible delayed hemolytic transfusion reaction.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A patient with a panreactive warm autoantibody received red cells 3 weeks ago. Which adsorption is used to look for underlying alloantibodies?

Autologous adsorption with the patient's own red cells

Incorrect. The sample still contains donor red cells from the recent transfusion. They can adsorb a true alloantibody along with the autoantibody, so the adsorbed plasma could look free of it.

Rabbit erythrocyte stroma (RESt) adsorption

Incorrect. RESt adsorbs selected cold autoantibodies and can also bind anti-B and anti-P1. A warm IgG autoantibody is removed with red cells.

Allogeneic adsorption with selected red cells

Correct. Donor cells from the recent transfusion can still circulate and would remove an alloantibody along with the autoantibody. Allogeneic cells, chosen so the set preserves detection of clinically significant specificities, remove the autoantibody and leave the adsorbed plasma ready for alloantibody testing.

Hint

Ask whose red cells are circulating in the patient 3 weeks after a transfusion.
An adsorbing cell removes every antibody whose antigen it carries.

Review Adsorption and elution

Problem 2 of 3 · Hints available

A patient receiving an anti-CD38 antibody is screened with dithiothreitol (DTT)-treated reagent cells, which removes the drug interference and destroys Kell antigens. The patient's K type is unknown. Which red-cell selection follows?

K-negative units, with the other selection rules applied

Correct. DTT destroys Kell antigens, so the treated-cell screen cannot detect anti-K, and AABB guidance calls for K-negative units unless the patient is known to be K-positive. Antibodies to other DTT-sensitive antigens, such as k, Yta, and Dombrock, can also be missed.

Units of unknown K type, since the treated screen was negative

Incorrect. The treated cells no longer carry Kell antigens, so a nonreactive screen cannot show whether anti-K is present.

Units negative for both K and k, since DTT destroys both

Incorrect. k is present on nearly all donor units, so k-negative red cells are kept for a patient with anti-k. After DTT testing, the guidance calls for K-negative units unless the patient is known to be K-positive.

Hint

List what DTT does to the reagent cells besides removing CD38.
Ask which antibody a treated cell can no longer detect.

Review Neutralization and thiol reagents

Antibody panel: + the cell carries the antigen, 0 it lacks it
Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	0	+	0	+	0	0
2	+	+	0	0	+	+	+	0	+	0	+	+	+	0	+	0
3	+	0	+	+	0	0	+	+	+	+	+	0	+	+	+	2+
4	0	+	0	+	+	0	+	0	+	0	+	+	+	0	+	0
5	0	0	+	+	+	0	+	+	0	+	+	+	0	+	+	1+
6	0	0	0	+	+	+	+	+	+	+	0	0	+	+	+	0
7	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
8	+	0	0	+	+	0	+	+	+	0	+	0	+	+	+	0
9	+	0	+	+	+	+	+	0	+	+	0	+	+	0	+	1+
10	0	0	0	+	+	0	+	+	0	0	+	+	0	0	+	0
11	0	0	0	+	+	0	+	0	+	+	0	0	+	+	0	0

Problem 3 of 3 · On your own

An eluate from a man's DAT-positive red cells is tested at the antiglobulin phase. It reacts with cells 3, 5 and 9 below. The last wash reacts 1+ with the same cells. What does the laboratory do with the eluate result?

Report anti-E eluted from his red cells

The last wash shows free anti-E left around the cells. That antibody reached the eluate by carryover, so the eluate cannot show that anti-E was bound.

Read an eluate without a nonreactive last wash

Unbound antibody left in the plasma around the cells can carry over into the eluate and react as though it had been eluted. A reactive last wash shows this carryover and invalidates the eluate. Without a nonreactive last wash, an antibody from the plasma could be wrongly reported as bound to the cells, so the last wash is tested in parallel with the eluate.

Subtract the last-wash grades from the eluate grades and report what remains

Reaction grades cannot be subtracted. A reactive last wash invalidates the whole eluate.

Call the eluate invalid, wash fresh cells until the last wash is nonreactive, and repeat the elution

Carryover makes this eluate uninterpretable. A repeat with adequate washing and a nonreactive last wash can show whether anti-E is bound to his cells.

Report the eluate as nonreactive, since the last wash cancels its reactions

The eluate did react. A reactive last wash makes that result uninterpretable, and the elution is repeated.

Review Adsorption and elution

Use it

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

Evelyn Coates, 68, a retired piano teacher (MRN 6204417), has multiple myeloma and receives daratumumab, an anti-CD38 drug.
She received 2 units of red cells 4 weeks ago at another hospital. No phenotype or genotype from before the drug is on file.
Today her plasma reacts 1+ with every screening and panel cell at the antiglobulin phase. Her autocontrol is nonreactive.
Her antibody screen with DTT-treated screening cells is nonreactive.
She needs 1 unit of red cells today.

Decision 1 of 3

What does the nonreactive DTT-treated screen show?

The reactions came from the drug, and antibodies to K and other DTT-sensitive antigens remain undetected

DTT removed CD38 and the drug's reactions with it. It also removed Kell antigens, so the treated cells could not show anti-K.

She has no clinically significant alloantibodies

The treated cells no longer carry K. Anti-K, and antibodies to other DTT-sensitive antigens, would not be detected by this screen.

Excluded anti-K using DTT-treated cells

Dithiothreitol (DTT) removes anti-CD38 interference by destroying CD38, and it also destroys Kell antigens on the treated cells. A nonreactive DTT-treated screen therefore cannot show that anti-K is absent, so K-negative units are given unless the patient is known to be K-positive.

DTT removed a warm autoantibody from her plasma

DTT treated the reagent cells, and her plasma was left untreated. Her nonreactive autocontrol also argues against an autoantibody.

Review Neutralization and thiol reagents

Decision 2 of 3

The service wants her extended red cell antigen profile to match future units. How is it obtained?

Serologic typing of today's specimen with antisera

Donor cells from 4 weeks ago are still in the specimen. A serologic type would test the mixture and could show antigens she lacks.

Typed a recipient's mixed red-cell population

After a recent transfusion, donor and patient red cells share the specimen, and a strong DAT can also distort serologic typing. The patient's antigen profile is determined by genotyping or by typing a separated patient-cell fraction, because a phenotype read from that mixture can show antigens the patient lacks and hide which alloantibodies the patient could form.

Serologic typing of her red cells after DTT treatment

DTT destroys Kell antigens on the treated cells, and the donor cells are still mixed in. Both problems remain.

Red cell genotyping from a blood specimen

Genotyping tests DNA from her white cells. Donor red cells have no nuclei, so they do not change the result.

Review Mixed red-cell populations and molecular testing

Decision 3 of 3

Her genotype predicts K+k+. What does that change for her units?

K-negative units are no longer required

She carries K and cannot make anti-K. The K-negative requirement after DTT testing applies to patients not known to be K-positive.

Nothing, because the DTT-treated screen still cannot detect anti-K

The DTT-treated screen cannot detect anti-K. A K-positive patient does not make anti-K, so that limitation no longer affects her unit selection.

The screen can now be done with untreated cells

The drug still binds CD38 on untreated cells. Every cell would react again.

Review Neutralization and thiol reagents

Two facts on her record directed this workup: daratumumab therapy and a transfusion 4 weeks ago. The drug explains the panreactivity and requires DTT-treated cells, which cannot detect anti-K. The recent transfusion makes serologic typing unreliable, so her antigen profile comes from genotyping.

For reference

Sources checked September 27, 2026

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Adsorption, elution, thiols and mixed fields

Try first

Get the idea

Autoantibody adsorption

Elution and the last wash

DTT and anti-CD38 drugs

Typing a mixed population

Watch one

Your turn

Use it

For reference

The rest of this step has a short briefing, a demonstration at the bench, 3 practice problems and a short case.

Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	+	+	0	0	+	2+
2	+	+	0	0	+	+	+	0	+	+	0	+	+	+	0	0
3	+	0	+	+	0	0	+	0	+	0	+	0	+	0	+	0
4	0	+	0	+	+	0	+	+	+	0	+	+	+	+	+	1+
5	0	0	+	+	+	0	+	0	+	+	+	+	0	+	+	0
6	0	0	0	+	+	+	+	0	+	+	0	0	+	0	+	0
7	0	0	0	+	+	0	+	+	0	0	+	+	+	+	+	2+
8	+	0	0	+	+	0	+	0	+	0	+	+	0	+	0	0
9	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
10	+	+	+	+	+	0	+	+	+	+	0	+	+	0	+	1+
11	0	0	0	+	+	0	+	+	+	+	+	0	+	+	+	1+

Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	0	+	0	+	0	0
2	+	+	0	0	+	+	+	0	+	0	+	+	+	0	+	0
3	+	0	+	+	0	0	+	+	+	+	+	0	+	+	+	2+
4	0	+	0	+	+	0	+	0	+	0	+	+	+	0	+	0
5	0	0	+	+	+	0	+	+	0	+	+	+	0	+	+	1+
6	0	0	0	+	+	+	+	+	+	+	0	0	+	+	+	0
7	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
8	+	0	0	+	+	0	+	+	+	0	+	0	+	+	+	0
9	+	0	+	+	+	+	+	0	+	+	0	+	+	0	+	1+
10	0	0	0	+	+	0	+	+	0	0	+	+	0	0	+	0
11	0	0	0	+	+	0	+	0	+	+	0	0	+	+	0	0

Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	+	+	0	0	+	2+
2	+	+	0	0	+	+	+	0	+	+	0	+	+	+	0	0
3	+	0	+	+	0	0	+	0	+	0	+	0	+	0	+	0
4	0	+	0	+	+	0	+	+	+	0	+	+	+	+	+	1+
5	0	0	+	+	+	0	+	0	+	+	+	+	0	+	+	0
6	0	0	0	+	+	+	+	0	+	+	0	0	+	0	+	0
7	0	0	0	+	+	0	+	+	0	0	+	+	+	+	+	2+
8	+	0	0	+	+	0	+	0	+	0	+	+	0	+	0	0
9	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
10	+	+	+	+	+	0	+	+	+	+	0	+	+	0	+	1+
11	0	0	0	+	+	0	+	+	+	+	+	0	+	+	+	1+

Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	0	+	0	+	0	0
2	+	+	0	0	+	+	+	0	+	0	+	+	+	0	+	0
3	+	0	+	+	0	0	+	+	+	+	+	0	+	+	+	2+
4	0	+	0	+	+	0	+	0	+	0	+	+	+	0	+	0
5	0	0	+	+	+	0	+	+	0	+	+	+	0	+	+	1+
6	0	0	0	+	+	+	+	+	+	+	0	0	+	+	+	0
7	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
8	+	0	0	+	+	0	+	+	+	0	+	0	+	+	+	0
9	+	0	+	+	+	+	+	0	+	+	0	+	+	0	+	1+
10	0	0	0	+	+	0	+	+	0	0	+	+	0	0	+	0
11	0	0	0	+	+	0	+	0	+	+	0	0	+	+	0	0

Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	+	+	0	0	+	2+
2	+	+	0	0	+	+	+	0	+	+	0	+	+	+	0	0
3	+	0	+	+	0	0	+	0	+	0	+	0	+	0	+	0
4	0	+	0	+	+	0	+	+	+	0	+	+	+	+	+	1+
5	0	0	+	+	+	0	+	0	+	+	+	+	0	+	+	0
6	0	0	0	+	+	+	+	0	+	+	0	0	+	0	+	0
7	0	0	0	+	+	0	+	+	0	0	+	+	+	+	+	2+
8	+	0	0	+	+	0	+	0	+	0	+	+	0	+	0	0
9	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
10	+	+	+	+	+	0	+	+	+	+	0	+	+	0	+	1+
11	0	0	0	+	+	0	+	+	+	+	+	0	+	+	+	1+

Cell	D	C	E	c	e	K	k	Fy^a	Fy^b	Jk^a	Jk^b	M	N	S	s	AHG
1	+	+	0	0	+	0	+	+	0	+	0	+	0	+	0	0
2	+	+	0	0	+	+	+	0	+	0	+	+	+	0	+	0
3	+	0	+	+	0	0	+	+	+	+	+	0	+	+	+	2+
4	0	+	0	+	+	0	+	0	+	0	+	+	+	0	+	0
5	0	0	+	+	+	0	+	+	0	+	+	+	0	+	+	1+
6	0	0	0	+	+	+	+	+	+	+	0	0	+	+	+	0
7	0	0	0	+	+	0	+	0	+	+	+	+	+	0	+	0
8	+	0	0	+	+	0	+	+	+	0	+	0	+	+	+	0
9	+	0	+	+	+	+	+	0	+	+	0	+	+	0	+	1+
10	0	0	0	+	+	0	+	+	0	0	+	+	0	0	+	0
11	0	0	0	+	+	0	+	0	+	+	0	0	+	+	0	0