Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

Abstract

In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients' sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

Figure 1.

IAT and DAT by FACS on AIHA samples characterized by the conventional (agglutination-based) IAT and DAT. (A) Results of IAT by FACS on samples in which IgM, IgG or IgM & IgG were detected by routine diagnostics, showing that the FACS-based method is less sensitive than the agglutination-based method especially for IgM. The number of patients tested per group is indicated in brackets. (B) Results of the FACS-based DAT on samples that were characterized as having IgG, IgG & C3, IgG & IgM & C3, IgM & C3 or C3 by routine diagnostic DAT (column method), showing strong similarity between the two techniques. The number of patients tested per group is indicated in brackets. (C) The FACS (figure) and diagnostic DAT (table) results are shown for a patient for whom the diagnostic DAT was problematic because of a positive phosphate-buffered saline control, but for whom the FACS result was very clearly only positive for IgM and C3 with nice negative controls.

Figure 2.

Testing methods to study complement activation by anti-RBC antibodies using ABO mismatching as a model system. (A) Group O serum was fractionated and fractions were tested for complement activation on AB RBC, and IgM/IgG concentration was measured. Complement activation can be seen to occur in the IgM-containing peak, as expected. (B) Group O serum was depleted of IgG or IgM and subsequently tested for complement activation on AB RBC. As can be seen, depleting group O serum of IgM strongly reduced the complement activation capacity, while IgG depletion left this ability intact, as expected for IgM-mediated complement activation.

Figure 3.

Identification of the antibody causing complement activation in AIHA. Sera from AIHA patients with different DAT results were fractionated and the fractions were tested for complement activation and IgG/IgM concentration. For each group of patients, complement activation was seen in the IgM-containing fractions. (A) DAT only positive for C3; (B) DAT positive for IgG, IgM and C3; (C) DAT positive for IgG and C3.

Figure 4.

Verification with an independent method that IgM is responsible for complement activation in AIHA. AIHA patients’ serum was depleted of IgG and IgM followed by a complement activation assay. As can be seen, IgM depletion strongly reduced complement activation whereas IgG depletion left this activity mostly intact. This verifies the fractionation results that anti-RBC IgM causes complement activation in AIHA. Diagnostic DAT results are presented in the tables.

Figure 5.

Identification of the complement-activating anti-RBC antibody in eluate versus plasma. Fractionation of eluate (A) and recalcified plasma (B) of an AIHA patient and subsequent testing of complement activating ability of the different fractions. As can be seen, complement activation in the plasma was IgM-mediated and in the eluate IgG-mediated. Diagnostic DAT results are presented in the table. No IgM was measurable in the eluate.

Figure 6.

Proposed diagnostic workflow for AIHA. The conclusion of the middle arm is based on the results presented in this paper.