Background: The direct antiglobulin test (DAT) identifies the presence of immunoglobulins and complement on the surface of red blood cells and is used to differentiate immune from non-immune hemolysis. Standard DATs done by conventional methods, using anti-IgG or anti-C3 antihuman globulin (AHG) in tube or gel, are performed routinely by laboratories associated with hospital transfusion services. However, if the standard DAT is negative but a strong clinical suspicion for immune-mediated hemolysis remains, serologic testing at an immunohematology reference lab (IRL) can be performed. Subtle differences in reagents and technique in the IRL may identify red cell-bound antibodies missed at the local hospital-based laboratory. In addition, the clinician can request an enhanced DAT, colloquially referred to as a Super Coombs, as a send-out test. This test can detect antibodies that are not detected by standard methods and that have been reported to cause autoimmune hemolysis, namely IgG bound at low levels, low affinity IgG, and IgA. We conducted a retrospective review of enhanced DATs sent by hematologists at the University of Pittsburgh Medical Center and the Allegheny Health Network to an outside IRL to determine the incidence of new autoantibodies detected by the enhanced DAT and assessed the impact of these results on patient management.Methods: We retrospectively identified patients at our center on whom enhanced DATs were sent from January 2019 to January 2021. Demographic, laboratory, and clinical data were collected and analyzed.Results: Twenty one patients were identified as having enhanced DATs sent to one outside IRL during the two year period. The majority of patients had abnormal clinical laboratory markers of hemolysis (81% with an elevated lactate dehydrogenase, 90% with an undetectable haptoglobin) and a history of immune dysregulation (79%). About 2/3 of the patients had a hemoglobin nadir above 6 g/dL. The median number of DATs performed locally prior to requesting the enhanced DAT evaluation was 2 (5 th to 95 th percentile: 1 - 3). Four out of 21 patients (19%) had previously tested positive at the local IRL by conventional serologic methods and 17 (81%) had tested negative.Among the 21 patients, 7 (33%) had at least one positive test on the enhanced DAT battery, including 5 out of the 17 (29%) who had previously tested negative locally. In 5 out of those 7 patients who tested positive by enhanced methods, the positive tests included standard serologic test done by conventional means. The only specialty serologic tests that yielded positive results was testing done at 4 °C, meant to detect low affinity IgG. The mean (±SD) overall turnaround time from sample collection to receipt of the enhanced DAT results was 3.8 (±1.5) days, the bulk of which was transit time from the local hospital to the outside IRL (2.5 (±1.5) days). Empiric high-dose steroid therapy was started in 68% of the patients prior to receipt of the enhanced DAT results. The median time between the first DAT result at the local IRL and the initiation of high dose steroid therapy was 1.5 (5 th to 95 th percentile: 0 - 12.5) days, while the median time from steroid initiation to the time the enhanced DAT was ordered was 5 (5 th to 95 th percentile: 1-256) days. Four patients were diagnosed with DAT-negative autoimmune hemolytic anemia based on response to steroids, whether complete (Hb ≥ 12 g/dL and normalization of LDH and haptoglobin) or partial (Hb improvement by 2 g/dL and no transfusion requirement), despite negative local and enhanced DAT results (table 1). Patients who had self-limited (2 patients) or subacute, non-severe anemia (5 patients) did not undergo a steroid trial and were diagnosed by non-serologic testing, specifically peripheral blood flow cytometry or bone marrow biopsy.Conclusions: The tests that most often yielded positive results on the enhanced DAT were standard serologic tests, suggesting that differences in timing of sample collection or technique may have explained result disparity between the local and outside IRL. Specialty testing done at 4 °C detects low affinity IgG antibodies bound to red cells, but the clinical significance of these antibodies is unclear. The majority of patients in our cohort underwent an empiric trial of high dose steroids prior to receipt of the enhanced DAT results. For these patients, the response to steroids was the deciding factor in AIHA diagnosis rather than the enhanced DAT results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.
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Open Access
Feb 13, 2024
Norma Bautista-Lopez + 1
