Jeremy W. Jacobs,T.D. Schlafer
The importance of the immunohematologic evaluation for presumed autoimmune hemolytic anemia in the COVID-19 era
The importance of the immunohematologic evaluation for presumed autoimmune hemolytic anemia in the COVID-19 era

Dear Editor, Daryani and colleagues recently described a pediatric patient with marked anemia in the setting of SARS-CoV-2 polymerase chain reaction (PCR) positivity. In this case, the authors effectively describe the laboratory evaluation, including the biochemical evidence of hemolytic anemia, in this patient. However, the only conclusive evidence for an autoimmune-mediated component is the positive direct antiglobulin test (DAT) using a poly-specific reagent and relatively non-specific peripheral blood smear morphology. Conversely, although autoimmune hemolytic anemia (AIHA) has classically been defined as hemolytic anemia with a positive DAT and concurrent exclusion of alternative diagnoses, the definitive diagnosis of immune-mediated hemolytic anemia, and particularly the subtype of AIHA (which dictates the treatment), requires further immunohematologic investigation. This is particularly true in the setting of SARS-CoV-2 infection due to the potential for non-specific DAT results. The DAT is simply designed to determine if immunoglobulin G (IgG) and/or complement (C3) are bound to an individual's own red blood cells (RBCs). A DAT positive for IgG can be secondary to various causes, including autoimmune conditions, drugs or intravenous immune globulin, and non-specific protein adsorption, such as in the setting of inflammation, among others. Furthermore, the strength of the DAT may not necessarily correlate with the presence or severity of hemolysis, and not all positive DATs are clinically significant. Prior studies have shown that up to 0.1% of healthy blood donors, 15% of critically ill patients, and up to 46% of patients with COVID-19 have positive DATs without evidence of AIHA. Thus, it is important to assess the DAT in the context of additional immunohematologic tests, including the antibody detection test (indirect antiglobulin test (IAT)), auto-control (reacting the patient's plasma with the patient's RBCs), and elution (removing the RBC bound immunoglobulins and determining their specificity). Moreover, the methodology employed to assess the DAT is critical, as various techniques have differing sensitivities. For example, in many hospital blood banks, column agglutination (i.e., gel methodology) is often much more sensitive than standard tube-based testing methods; thus, while gel-based techniques may detect more positive DATs, these results may or may not reflect clinically significant autoantibodies. This highlights the importance of reporting the testing methodology employed, as well as further confirmation of the specificity and clinical significance of the autoantibody via elution studies when IgG antibodies are bound to RBCs. While we do not necessarily disagree with the authors’ assertion that this patient developed hemolytic anemia in the setting of COVID-19, we believe it is important to highlight the importance of immunohematologic evaluation to confirm a diagnosis. Conversely, we do question the specific diagnosis of mixed AIHA, as this particular subtype of AIHA is characterized by the combination of warm autoantibodies (typically IgG) and cold autoantibodies (typically IgM, which fix C3). To definitively diagnose mixed AIHA requires demonstration of a warm autoantibody, classically associated with a DAT positive for IgG and a pan-reactive eluate, as well as a cold autoantibody, classically associated with a DAT positive for C3. Furthermore, as certain IgG isotypes can bind complement, diagnosing mixed AIHA generally requires demonstration of a cold agglutinin at high titer (>1:64) with broad thermal amplitude (reactive at ≥28–30 °C). Given that the authors did not perform, or at least did not report, this testing, the diagnosis of AIHA, and particularly mixed AIHA, is presumptive at best. Finally, while the authors astutely comment on the rarity of SARS-CoV-2-associated AIHA in the pediatric population, we previously described five pediatric patients in a systematic review with AIHA in the setting of SARS-CoV-2 infection. Among these patients, two had warm AIHA, one had mixed AIHA, one had cold AIHA, and one had Evans syndrome, with the subtype of AIHA not specified. Thus, we agree with the authors that healthcare providers should be cognizant of the potential for SARS-CoV-2 to mediate AIHA, even in the pediatric population. While we commend the authors for highlighting the potential adverse events associated with SARS-CoV-2 infection, we caution readers and authors from drawing definitive conclusions in the absence of complete laboratory evaluation. As misinformation surrounding COVID-19 and SARS-CoV-2 vaccines designed to protect individuals abound, , we believe that researchers and physicians should ensure that thorough laboratory assessment be performed before reporting potential complications secondary to infection and vaccination.

The authors have none to declare.