Hemoglobin versus ADAMTS13

Comment on Studt et al, page 542

Free hemoglobin inhibits ADAMTS13 activity in vitro, demonstrating a potential source of artifact affecting ADAMTS13 activity assays and inhibitor screens.

Deficiency of the ADAMTS13 metalloprotease causes thrombotic thrombocytopenic purpura (TTP). The rare familial form of TTP results from mutations in the ADAMTS13 gene that lead to loss of plasma ADAMTS13 activity.¹  In contrast, the more common acquired form of TTP is an autoimmune disorder characterized by neutralizing autoantibodies directed against ADAMTS13 that inhibit its activity.^2,3  Although the ability to determine ADAMTS13 activity and perform anti-ADAMTS13 antibody screening would be of great diagnostic value for the clinician, the use of these tests currently is restricted by limited availability and long turnaround times. Therefore the clinical application of ADAMTS13 testing in the management of TTP is still evolving, and continues to be the subject of considerable research activity.

In this issue of Blood, Studt and colleagues describe a case of familial TTP that led to an unexpected and important observation: free hemoglobin inhibits ADAMTS13 activity in vitro. The patient was a 7-year-old boy who suffered from repeated episodes of thrombocytopenia and Coombs-negative hemolytic anemia, and had been given the diagnosis of atypical Evans syndrome. Unfortunately the child died of a severe attack of his disease, but autopsy was suggestive of TTP. The diagnosis of familial TTP was established subsequently by ADAMTS13 activity assay (< 3% in the patient, and ∼50% in each parent) and ADAMTS13 sequencing (the patient was homozygous for a splice acceptor site mutation, and each parent was heterozygous).

During the course of the workup, the authors noticed that the patient's serum inhibited ADAMTS13 activity in normal plasma, suggesting the presence of anti-ADAMTS13 inhibitory antibodies. However, purified total immunoglobulin (Ig) had no inhibitory effect, while Ig-depleted serum still inhibited ADAMTS13 activity. Together these results indicated that the inhibitory capacity of the patient's serum was due to factors other than anti-ADAMTS13 antibodies. As the sample was highly hemolyzed, the authors hypothesized that products of hemolysis may be responsible for the inhibitory activity they were observing. This hypothesis turned out to be correct, as the authors went on to demonstrate that free hemoglobin (either recombinant or from lysed erythrocytes) inhibits ADAMTS13 activity in normal plasma. This finding was replicated in 3 other laboratories, each using different ADAMTS13 activity assays.

Several important conclusions can be drawn from this interesting report. First, as the authors point out, the fatal course of the child misdiagnosed with Evans syndrome emphasizes the need for rapid diagnostic testing for patients suffering from possible congenital thrombotic microangiopathies, as plasma therapy is highly effective and can be lifesaving. Second, the presence of hemoglobin (as can result from in vitro hemolysis) may influence many of the available ADAMTS13 activity assays. Therefore caution should be used under these circumstances lest the incorrect assumption of decreased ADAMTS13 activity or presence of inhibitory anti-ADAMTS13 antibodies be made. Finally, this case is an excellent example of how thorough investigation of a surprising observation can yield important information, and further illustrates the importance of the interface between clinical medicine and basic research. ▪