15.3 Adsorption, Elution, Titration, and Cell Separations

15.3 Adsorption, Elution, Titration, and Cell Separations

Quick Answer: Autoadsorption removes autoantibody onto the patient’s own cells so you can look for an alloantibody — do not autoadsorb if the patient was transfused in the last 3 months. Use allogeneic differential adsorption when donor cells are still circulating or autologous cells are insufficient. RESt strips most cold autoantibodies. Elution recovers in-vivo coating: acid for warm IgG, Lui freeze-thaw (or heat) for ABO. Test the eluate and a last-wash control. Titers for HDFN are AHG titers; compare the current specimen with the previous one in parallel. After transfusion, hypotonic / retic enrichment or microhematocrit separation lets you phenotype the patient’s own young cells.

The June 9, 2026 outline groups these under IV.C. They are not decoration around a panel. They are how you finish a warm auto, a DAT-positive hemolyzing newborn, a rising prenatal anti-D, and a phenotype on somebody who was transfused yesterday.

Autoadsorption — and the three-month rule

A warm autoantibody coats the patient’s cells and usually reacts with every panel cell. You cannot tell whether an alloantibody is hiding underneath until you remove the auto from the plasma.

Autoadsorption incubates the patient’s plasma with the patient’s own red cells (often pretreated so more auto binds). The auto goes onto the cells. The adsorbed plasma is then screened and paneled for leftover alloantibody.

Do not autoadsorb if the patient was transfused in the last 3 months. Circulating donor red cells are still there. Those cells can adsorb an alloantibody directed at a donor antigen the patient lacks. You will then report “auto only, no alloantibody,” issue antigen-positive blood, and cause a delayed hemolytic reaction. That three-month window is an outline-level contraindication, not a suggestion.

Other reasons autoadsorption fails: the patient is too anemic to spare cells, the auto is so strong that residual antibody remains after several passes, or you forgot that enzyme- or ZZAP-pretreated autologous cells are used to strip coating IgG and improve uptake — those pretreatments also destroy some antigens (Duffy, MNS, Kell with ZZAP), so you cannot read the adsorbed plasma as “the patient has no Duffy antibody” just because you used ZZAP-treated cells. Thiol and ZZAP details belong in the next chapter; the adsorption rule here is know what you destroyed before you interpret silence.

Cold autoadsorption at 4 °C is the analogous move for a cold agglutinin when you still have autologous cells and no recent transfusion.

Allogeneic differential adsorption

When autoadsorption is forbidden or impossible, adsorb aliquots of plasma onto selected allogeneic cells of known phenotype — classically a set such as R1R1, R2R2, and rr cells that also differ in K, Jk, Fy, and S. Each aliquot removes antibody to antigens present on that cell and leaves antibody to antigens that cell lacks.

Compare the three leftover specificities. Anti-Jka survives only on the Jk(a−) adsorbing cell. Anti-E survives on the R1R1 (E−) cell and disappears on R2R2. That is differential adsorption: the pattern of what each cell stole is the identification.

Use allogeneic adsorption for:

• Transfusion in the last 3 months
• Insufficient autologous cells
• Separating a mixture of alloantibodies when a straight panel is stuck

Do not adsorb with random outdated units of unknown phenotype and call it differential.

RESt for colds

Rabbit erythrocyte stroma (RESt) is packed with I/IH/H-like structures. Incubating plasma with RESt removes most cold autoantibodies (anti-I, anti-IH, anti-H) so you can finish an ABO reverse type or a 37 °C screen.

RESt is not a specific anti-I sponge. It can also remove anti-B, anti-P1, and, in published cases, anti-D, anti-E, or anti-Vel. After RESt, do not announce that those specificities were never there. If the reverse type still needs anti-B, confirm with washed A1/B cells, prewarm, or a different cold-removal method rather than assuming RESt is inert.

Elution: get the coating antibody off the cell

An eluate is the antibody you recovered from DAT-positive red cells. You identify it the same way you identify a plasma antibody — against a panel, and against A and B cells when ABO is in play.

Lui freeze-thaw is the method the exam still pairs with ABO HDFN. Acid elution is what you reach for when the DAT is IgG-positive in a warm auto or a delayed Kidd reaction.

Last-wash control: the saline (or working wash) from the final wash before elution is tested in parallel with the eluate. It must be nonreactive. If the last wash still agglutinates panel cells, you never removed unbound plasma antibody, and the “eluate” is contaminated plasma. Repeat the washes; do not identify a phantom coating antibody.

Test the eluate against screening or panel cells and, when the patient is a group A or B neonate of a group O mother, against A1 and B cells. An eluate that reacts only with A cells is anti-A, even if the plasma antibody screen is negative.

Titration

A titer is the reciprocal of the highest dilution that still gives the SOP endpoint, usually 1+. Prepare a master serial twofold dilution of plasma and test every tube against the same antigen-positive cells.

• Saline / immediate-spin titers measure IgM agglutinins (ABO isoagglutinin studies, some colds). They do not manage HDFN.
• AHG titers measure IgG. That is the prenatal titer.

Compare the current specimen with the previous specimen in parallel — same day, same cell phenotype, same method, same endpoint. A one-tube bump can be noise. A two-tube (fourfold) rise is a significant change. Scoring (4+ = 12, 3+ = 10, 2+ = 8, 1+ = 5) can show a real increase when the endpoint tube has not moved yet.

Critical prenatal titers are method-dependent. Many U.S. tube-AHG laboratories use 16 (some use 32) as the threshold that triggers MCA-PSV Doppler. Gel/column titers run higher, so a gel 16 is not a tube 16. Anti-K does not wait for that number — Chapter 12 already made that exception. Do not titer Lewis, P1, or other 37 °C-negative nuisances and call it an HDFN workup.

Passive anti-D after antenatal RhIG is usually low (≤4, sometimes 8) and flat. A rising AHG titer is immune anti-D, not leftover RhIG.

Cell separations after transfusion

A recently transfused sample is a mixture. Antigen typing that sample is typing the donors plus the patient. Mixed-field typing is a clue, not a phenotype.

Reticulocytes and young red cells are the patient’s own product (unless erythropoiesis is shut down). They are less dense than mature donor cells.

• Microhematocrit centrifugation: spin, harvest the top (least dense) layer, and type those cells.
• Hypotonic / commercial retic enrichment: mature cells lyse or pellet preferentially; the retained young-cell fraction is enriched for autologous antigen.

Use the separated cells for extended phenotyping when you need antigen-negative units and the pretransfusion type is unknown. If the patient was massively transfused or is not making retics, separation lies — go to molecular typing (Section 15.4).

Do not issue a full phenotype from an unseparated EDTA drawn three days after four units and call it the patient’s Kell/Duffy/Kidd type.

Which method when

Exam traps

• Autoadsorbing a patient transfused last month.
• Skipping the last-wash and identifying leftover plasma as an eluate.
• Using acid elution as the only named method for ABO HDFN when the stem wants Lui freeze-thaw.
• Titering this month’s specimen alone and calling a one-tube change significant.
• Phenotyping an unseparated post-transfusion sample.
• Treating RESt as incapable of removing anything except anti-I.