Role of Eluate Testing in the Diagnosis of Immune-Mediated Hemolytic Anemias with Positive Direct Antiglobulin Tests
Authors: Bikkani Thejaswi , Weinheimer Kaitlyn D , Ruiz Lopez Jorge N , Panch Sandhya , Hasan Rida 
Published: December 2024

Background:
Direct antiglobulin testing (DAT) is routinely ordered to evaluate for immune-mediated hemolytic anemia. Elution studies (surface antibody separation) may also be performed with positive DATs to further characterize the autoantibody specificity. A positive DAT but a negative eluate is classically thought to be suggestive of a drug-dependent antibody, hypergammaglobulinemia, or an antibody against a low-incidence antigen. A negative eluate is not typically indicative of a clinically significant warm or cold autoimmune hemolytic anemia (WAIHA or CAD). Elution studies are not always performed following positive DATs and the interpretation of negative DATs can vary. We evaluated our institutional data to determine patterns of elution testing in patients with positive DATs who were evaluated for WAIHA and/or CAD.
Methods:
This is a retrospective, single-center review of all positive DATs at our institution from April 2021 to April 2024 clinician ordered DATs. We do not have a standardized protocol for when to perform elution studies and this testing can be requested at the discretion of the medical director. If the IgG monospecific testing is negative, then we do not usually perform elution studies.
We collected data on the strength of the DAT (strength of reactions, elution studies), antibody screen results, laboratory data pertinent to the evaluation of hemolysis (hemoglobin, absolute reticulocyte count within the last 48 hours as well as LDH, haptoglobin, and bilirubin within the last week). We then reviewed charts to determine if the patient was diagnosed with an immune-mediated hemolytic anemia, type of anemia, secondary causes and treatment. T-tests and Mann-Whitney tests were used to compare the WAIHA patients with positive eluate and negative eluate using GraphPad prism.
Results:
We found 191 individual patients who had positive DATs ordered by a provider for evaluation for hemolysis. Of these patients, 43 (23%) had a clinical diagnosis of a hemolytic anemia. Within this group, 10 had a positive eluate (23%), 19 had a negative eluate (43%), 14 had no elution studies performed (34%). All positive eluates (n=10) showed a panagglutinin pattern consistent with a diagnosis of AIHA.
In the negative eluate group with a clinical diagnosis of hemolytic anemia (n=19), the etiologies were WAIHA (n=10), CAD (n=2) and nonspecific hemolytic anemia (n=7). 6 patients had primary diagnoses of autoimmune diseases. None of the patients with negative eluates had concerns for drug-dependent antibodies or antibodies against low-incidence antigens. In the no elution group with a clinical diagnosis of hemolytic anemia (n=14), the etiologies were WAIHA (n=6), cold agglutinin disease (n=3), nonspecific hemolytic anemia (n=5).
T-test compared WAIHA patients with positive and negative eluates. Positive eluate group had higher strength on polyspecific DAT (2.9+ versus 1.7+, p=0.0013) and on IgG strength (3+ vs 1+, p=0.004). There were no differences in lowest hemoglobin 48 hours prior (7.2g/dL vs 8.0g/dL, p=0.35), highest reticulocyte percentage 48 hours prior (2.3% vs 2.6%, p=0.49), highest indirect bilirubin 7 days prior (1.5mg/dL vs 2.3mg/dL, p=0.59) between the positive and the negative eluate groups. The calculated positive and negative predictive values for eluates in making a diagnosis of AIHA were 38% and 84%, respectively.
Conclusion:
In patients with concerns for clinical hemolysis and a positive DAT, 1 in 5 may have a confirmed diagnosis of immune-mediated hemolytic anemias. Elution studies, when positive can aid in the diagnosis of AIHA. Negative elution studies do not rule out AIHA but may require that clinicians consider other diagnoses, including drug-dependent antibodies as the cause for AIHA. AIHA with negative elution studies have decreased strength on testing but their clinical hemolysis labs are not significantly different than those with positive eluates.
Clinician understanding of DAT testing protocols are critical to make accurate diagnoses of immune-mediated hemolytic anemias. Additional studies are ongoing to evaluate response to therapy and outcomes in the various categories of AIHA above as a way to further streamline management of these patients.