Warm-Reactive Autoantibodies - Confirmation: Eluate Testing

Once a warm autoantibody is detected, a direct antiglobulin test (DAT) should be performed (see Module 1 for additional details on this test). The DAT will typically be positive for IgG but may be positive for C3 as well.

  • DAT-negative autoimmune hemolysis: Approximately 5-10% of cases of autoimmune hemolytic anemia will have a negative direct antiglobulin test. This situation can arise due to low titer or low affinity antibodies, or the occurrence of unusual antibody classes such as IgA. Proving the presence of an autoantibody in these cases requires specialized testing protocols with enhanced sensitivity (e.g. flow cytometry, anti-IgA antiglobulin reagent, etc.)

Further confirmation that a DAT is positive due to a warm autoantibody can be obtained by eluting the antibody off the patient’s RBCs and then testing that eluate against panel cells by IAT. If the eluate is also shown to be a panagglutinin, then a warm autoantibody can be confirmed.

  • Performing an eluate: The first step in an elution is to wash the RBCs to remove any unbound immunoglobulins. Following this, dissociation of bound antibody can be accomplished by a variety of elution techniques:
    • Temperature: application of cold or heat (56ºC).
    • pH: use of acid reagents to alter the charge of the protein, and thus its configuration.
    • Organic solvents, which can denature the RBC membrane.
  • The choice of elution technique depends on the type of antibody you expect to elute. For example, freeze-thaw and heat elutions are used in case of ABO HDFN to elute anti-A and anti-B from neonatal red blood cells. Acid elution and ether elution are used to elute warm auto- or allo-antibodies. The most frequently used elution technique is the acid elution and is available in commercial kits.

Occasionally, however, the eluate will give unexpected results. If the eluate reacts with only some reagent cells but not others (ie., if it shows specificity for certain RBC antigens), that is more consistent with an alloantibody, such as may occur following the transfusion of incompatible RBCs.

Alloantibodies may also be eluted in cases of hemolytic disease of the fetus and newborn (in which neonatal RBC are bound with maternal antibodies) or passenger lymphocyte syndrome (in which B-cells co-infused during stem cell or solid organ transplantation secrete antibodies against the host antigen).

In rare cases, a patient will develop an autoantibody with specificity against a specific RBC antigen. Distinguishing these cases from alloantibodies requires demonstrating that the patient themselves expresses this same antigen. This may be performed by phenotyping. However, when attempting to distinguish alloantibodies from autoantibodies, genotyping has several advantages over phenotyping:

  • Phenotyping can be confounded by the presence of recently transfused RBCs and should be interpreted with caution if the patient has been transfused in the previous 3 months.
  • When using phenotyping reagents that rely on indirect antiglobulin testing, testing cannot be performed on RBCs that already have IgG on their surface (i.e.., patients with positive DATs).
  • Phenotyping may fail to identify patients with partial antigens (e.g., RhD or RHCE variants).

If the eluate fails to react with any panel cells at all, several possibilities should be considered:

  • The first is that the patient’s RBCs have been bound by anti-A or anti-B, as may occur following a non-ABO-identical platelet transfusion or an IVIG infusion. As the reagent cells used for antibody investigations are usually group O, isoagglutinins will not be detected in the eluate. Testing the eluate against cells of the patient’s own ABO group will help identify these isoagglutinins.
  • The second possibility is that the patient has developed an alloantibody against a very low-frequency antigen that was present on recently transfused RBCs, but not on the panel cells used to investigate the eluate. Re-crossmatching patient plasma against retained segments from previously transfused RBCs may detect such cases.
  • The third and most common explanation for a positive DAT with a non-reactive eluate is that the patient has developed an immune response against a drug which has subsequently bound to their RBCs; as the drug is lost or altered during the elution process, the eluted antibody will not bind to reagent cells unless they, too, have been pre-treated with the drug in question.

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