Preventing misphenotyping in a patient with warm autoantibody interference: A case report

Warm autoantibodies (WAA) are immunoglobulin G (IgG) antibodies that react optimally at 37ºC and pose a significant challenge in immunohematology due to their effect in routine serologic testing. These antibodies may cause autoimmune hemolytic anemia (AIHA) by coating red blood cells and activating complement systems. Their presence interferes with normal antigen-antibody interactions by disrupting antigen recognition thereby making accurate phenotyping difficult and complicating transfusion decisions.¹

Given the diagnostic pitfalls associated with WAA, especially in patients with a positive direct antiglobulin test (DAT), careful selection of testing methods becomes critical. This case illustrates the difficulties that arise when conventional red cell phenotyping is performed in the presence of WAA and underscores the value of integrating molecular genotyping as a confirmatory approach when serologic results are unreliable.

Case presentation

A 23-year-old female undergoing a transfusion workup for anemia of unknown origin presented with a strongly positive direct antiglobulin test (DAT) using polyspecific antihuman globulin, anti-IgG, and anti-C3 reagents. She had no history of transfusion within the past six months, reducing the likelihood of recent alloantibody stimulation. The autocontrol was reactive, and the antibody screen, saline panel, and eluate (Immucor Gamma ELU-KIT II) both demonstrated panreactivity, indicating the presence of a warm autoantibody (WAA). Antibody identification panels showed only autoantibody reactivity, excluding underlying alloantibodies. In addition, the patient had no transfusion history in the past three months, ruling out recent antigen exposure. Despite the known WAA interference, serologic phenotyping using conventional antisera for Rh (C, c, E, e) and Kell (K) antigens was attempted without any pretreatment of the red cells. All tested antigens appeared positive, suggesting a panreactive Rh and Kell phenotype (Table 1). To resolve the discrepancy, the sample was sent to the immunohematology reference laboratory (IRL) for molecular red cell genotyping within 24 hours. Genotyping results confirmed that patient was negative for e and Kell antigens, directly contradicting the serologic phenotype and confirming autoantibody interference as the cause of the false positives.

Discussion

Accurate red cell phenotyping is crucial for transfusion safety as it ensures that patients receive compatible blood, reducing the risk of hemolytic transfusion reactions.² However, WAA frequently interfere with conventional serologic phenotyping by masking or mimicking true antigen expression leading to erroneous results. This interference is primarily due to IgG coating of red cells reacting nonspecifically with anti-IgG reagents causing erroneous results that can mislead clinicians, and compromise patient safety and care efficacy.³

This case highlights a preventable diagnostic error resulting from unmodified serologic phenotyping in a DAT-positive sample. The patient’s red cells were heavily coated with IgG, and any serologic typing performed under these conditions is at risk for inaccuracy. The pattern of results including panreactivity, positive DAT, and uniform antigen reactivity, were incompatible with known antigen distribution, and increased the concern of spurious phenotyping results. These concerns are consistent with the known limitations described in the Instructions for Use (IFU) for commercial phenotyping reagents, which caution against interpreting results on DAT-positive red cells. Anti-IgG–based reagents may nonspecifically react with IgG coating resulting in agglutination that mimics true antigen expression. Conversely, IgM-based reagents, though commonly used, may still yield misleading reactions due to spontaneous agglutination or altered membrane integrity in WAA cases.³

In warm autoimmune hemolytic anemia, IgG autoantibodies coat red cell membranes, occupying antigenic sites or causing steric hindrance that prevents accurate antigen–antibody interaction with commercial antisera. This may yield false-positive or indeterminate reactions, particularly with low-affinity or enzyme-sensitive antigens. Failure to recognize this risk could result in inappropriate transfusion, alloimmunization, and, in pregnant patients, hemolytic disease of the fetus and newborn (HDFN).⁴

Several pretreatment strategies are available to minimize warm autoantibody interference. Warm saline washes can reduce nonspecific interference but are not sufficient in removing membrane-bound IgG.^5,6 Dithiothreitol (DTT) removes IgM but destroys Kell antigens, limiting its utility when Kell typing is required.^6,7 EDTA–glycine–acid (EGA) treatment works efficiently by striping IgG from red cells but similarly denatures Kell antigens, and may affect other labile antigens. Chloroquine diphosphate (CDP) is generally preferred because it effectively removes IgG while preserving most clinically significant antigen systems, though it may weaken Bg^a antigens.^5,6 Each pretreatment method exerts variable effects on antigen integrity. Warm saline preserves all major antigen systems but is least effective in removing IgG. DTT and EGA destroy disulfide bond–dependent Kell antigens, whereas chloroquine preserves Rh, Kidd, Duffy, and most MNS antigens, making it the preferred reagent when Kell typing is required.^5-7

Given these differences, failure to pre-treat the red cells to remove the autoantibody coating before phenotyping led to misinterpretation. Preventing such misinterpretation would require the use of validated red cell pretreatment and chemical methods.⁴ Early incorporation of molecular genotyping that ultimately provided clarity in this case, should be considered early especially when warm autoantibody interference is suspected. Molecular genotyping not only confirms serologic discrepancies but also enables extended antigen profiling in multi-transfused or chronically anemic patients where mixed-field agglutination may obscure true phenotypes.^9,10

Conclusion

Recognizing the limitations of serologic phenotyping in DAT-positive patients is critically important, particularly for those with warm autoantibodies. It highlights the importance of evaluating the serologic results carefully, adhering to validated pretreatment protocols to ensure accurate antigen typing, and integrating molecular methods for determining red cell phenotype when serologic testing is compromised. Failure to apply appropriate testing strategies may lead to significant misphenotyping, increasing the risk of alloimmunization and mismatched transfusions.

References

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