Class I HLA-Specific Antibodies Can Cause “false-positive” Test Results in the Serotonin Release Assay for Heparin-Induced Thrombocytopenia (HIT).

Abstract 3506

Poster Board III-443

A diagnosis of heparin-induced thrombocytopenia (HIT) is confirmed with support from the laboratory. Antibodies associated with HIT are specific for complexes made up of heparin and platelet factor 4 (PF4) and can be detected in a solid phase ELISA (PF4 ELISA). However, a functional test, the serotonin release assay (SRA) is regarded by many to be the “gold standard” for laboratory investigation of this disorder. In our facility, the SRA is performed by incubating serotonin-labeled platelets (pooled from three different group O donors) with test serum and low dose (0.1 units/ml) or high dose (100 units/ml) heparin and determining the percentage of total serotonin released. Release of serotonin (20-100%) with low dose heparin and inhibition of this release with high dose heparin is considered to be “positive” for platelet-activating HIT antibodies. Sera from some patients cause serotonin release with low dose heparin that is not inhibited with high dose heparin. The significance for these “indeterminate” reactions is unclear, but they are considered not to reflect the presence of “true” HIT antibodies.

We studied selected serum samples from 238 patients referred for HIT testing. Of these, 119 tested “true” positive and 117 produced “indeterminate” reactions in the SRA. The same samples were tested for the presence of antibodies reactive with beads coated with various Class I HLA antigens using a flow cytometric bead assay (Flow PRA, One Lambda). Sera producing at least 30% release in SRA and reactive with at least 20% of the bead panel were selected for analysis. As shown in Figure 1, there was a high correlation between the likelihood of an “indeterminate” SRA test result and the presence of Class I HLA antibodies (p << 0.0001).

As expected, there was a significant correlation between the strength of reactions produced by individual “true positive” sera in the SRA (% release) and in the PF4/heparin ELISA (O.D. value). However, in analyzing 38 sera with “true positive” test results in the SRA, we identified two that were negative in the PF4 ELISA and contained broad Class I HLA reactivity (reactive with 100% and 41% of the panel, respectively).

We conclude 1) Class I HLA antibodies are the major cause of “indeterminate” reactions in the serotonin release assay and 2) A subset of these antibodies can be inhibited by high dose heparin and therefore mimics the behavior of “true” HIT antibodies in the SRA. Unless the PF4 ELISA test is used together with the SRA, this type of reaction could lead to an erroneous diagnosis of HIT.