Serologic Characterization of Antibodies in Patients Experiencing Piperacillin-Induced Immune Thrombocytopenia
Authors: Rasmussen Mark , Bougie Daniel W. , Denomme Gregory H. , Aster Richard H. 
Published: November 2019

Beta lactam antibiotics (penicillins, cephalosporins, etc) are relatively common triggers for immune hemolytic anemia, neutropenia and drug-induced thrombocytopenia (DITP) but the mechanism(s) responsible for this side effect are not well understood. The ureidopenicillin, piperacillin, is one of the beta lactam drugs implicated most often as a trigger for immune cytopenia. We characterized drug-dependent reactions of antibodies (abs) identified in 18 patients with piperacillin-associated thrombocytopenia.
Each of the 18 patients had an ab that reacted strongly (flow cytometry) with normal platelets but not RBC when soluble piperacillin was present. These reactions were not inhibited by the highest drug concentration that could be achieved in the reaction mixture and similar antibodies were not found in normal serum. These reactions are similar to those obtained with drug-dependent antibodies (DDAbs) found in patients sensitive to quinine, vancomycin, and many other drugs known to cause DITP. Evidence suggests that such drugs promote binding of DDAbs to their targets by reacting with antibody CDR3 and modifying its specificity (Blood 2015;126:2138).
Beta-lactam drugs differ from most other medications in their ability to spontaneously link covalently to free amino groups on membrane proteins to produce potentially immunogenic haptens that could induce abs theoretically capable of contributing to thrombocytopenia in piperacillin-treated patients. We optimized conditions for "haptenization" of platelets and RBCs with piperacillin and tested patient and normal sera for abs that recognize piperacillin-coated cells. Complete inhibition of binding by excess soluble drug was a criterion for a "positive" reaction. As shown in Table 1, IgG and IgM abs reactive with piperacillin-coated RBCs were found in each of 18 patient and 20 normal sera tested; IgM abs reactive with piperacillin-coated platelets were found in nearly all of both groups and similar IgG abs were found in about half. Reaction strength of IgM abs against piperacillin-coated RBCs correlated closely with that against piperacillin-coated platelets
The findings demonstrate two distinctly different types of piperacillin-specific abs in patients experiencing piperacillin-induced thrombocytopenia. The first is usually IgG, binds to platelets but not RBCs only when soluble drug is present, is not inhibited by excess drug, even at high concentrations, correlates with exposure to piperacillin and development of thrombocytopenia and is not found in normal persons. This behavior is similar to that of DDAbs induced by quinine, vancomycin and many other drugs. The second is commonly IgM and less often IgG, binds to piperacillin-coated platelets and RBCs, is inhibited by soluble drug and, as the IgM isoform, is found in nearly all normal subjects. Failure of abs that recognize piperacillin-coated cells to distinguish between platelets and RBCs in any consistent way argues against the possibility that they play a role in the pathogenesis of piperacillin-induced DITP. "Naturally-occurring" IgM abs that recognize piperacillin-coated RBC were previously described by Garratty et al (Transfusion 2008;48:2429) and could reflect widespread environmental exposure to beta-lactam antibiotics.

Disclosures
No relevant conflicts of interest to declare.