Background/Case Studies: The direct antiglobulin test (DAT) is a serologic test to demonstrate the in-vivo coating of RBCs with immunoglobulin and/or complement. It investigates if a hemolytic process is due to an immune or nonimmune etiology. The DAT is used in diagnosing autoimmune hemolytic anemia (AIHA), an acquired immunologic disease resulting in RBC destruction by autoantibodies. However, some patients exhibiting clinical signs of AIHA may present with a negative DAT. For these patients, a DAT-negative investigation has been helpful in diagnosing AIHA.
Study
Design/Methods: The DAT-negative investigation includes multiple tests on fresh patient RBCs collected in an EDTA tube. These tests include a differential DAT using polyspecific anti-human globulin and monospecific anti-IgG and anti-C3b, as well as a saline control with patient RBCs tested at room temperature (RT) and with 4⁰C saline-washed patient RBCs. Additionally, a DAT is tested using a polyspecific gel card, and enhanced autocontrols (washed and unwashed RBCs) are tested at RT by indirect antiglobulin test using PEG and polybrene. An antibody screen is performed at the following phases: immediate spin, 37⁰C-LISS, LISS-IgG-AGT, PEG-IgG-AGT, and Ficin-IgG-AGT. An eluate is prepared and concentrated using a multi-well ultrafiltration sample concentrator. Lastly, DATs are performed using the patient RBCs tested against anti-IgG, anti-IgM, and anti-IgA in capillary tubes. A review and analysis were conducted on DAT-negative investigations performed at a high-volume immunohematology reference laboratory from 2021 to 2022.
Results/Findings: Over a two-year period, 148 DAT-negative investigations were performed; 58 in 2021 and 90 in 2022. Of the samples tested, 95 (64%) yielded a positive result. Table 1 outlines the test results, while a detailed breakdown of the positive results is provided to specify the test(s) in which patient plasma exhibited reactivity. Conclusions: The DAT-negative investigation is a valuable diagnostic tool for identifying AIHA when the conventional DAT test produces negative results. The laboratory findings in Table 1 demonstrated that AIHA was supported in about two-thirds of the samples that underwent the DAT-negative investigation. Therefore, it is highly recommended to conduct this investigation when there is suspicion of AIHA, but a routine DAT does not confirm the diagnosis.
Importance of research: Patients exhibiting clinical signs of AIHA may present with a negative DAT. For these patients, a DAT negative investigation has been helpful in diagnosing AIHA. This is a battery of more sensitive tests to determine is RBC are coated with immunoglobulin or complement.
