Platelet Proteins Cause Allergic Transfusion Reactions through an Immunoglobulin-Dependent Mechanism

Background: A general understanding of allergic transfusion reaction (ATR) mechanisms remains elusive. Various hypotheses invoke proteins, small molecules, mitochondria, or microparticles that may be plasma or platelet derived and suggest antibody dependent or independent mechanisms. There has been no systematic comparison of these proposed mechanisms. The aim of this study is to characterize the mechanistic determinants of ATRs.

Methods: Basophil enriched cell suspensions were collected from healthy donors (n=8). Basophil histamine release was measured in response to platelet-derived components: platelet component supernatant (plasma), platelet lysate, and manipulated platelet lysates were examined to characterize the unknown allergic stimuli. Lysate manipulations were: 1) dialysis against a 3,500 molecular weight cutoff membrane, 2) butanol/DIPE delipidation, 3) trypsinization with immobilized trypsin beads, 4) mild heat denaturation at 47˚C for 20 minutes, and 5) ultracentrifugation at 200,000 gfor 1hr. Immunoglobulin-dependent mechanisms were investigated through lactic acid immunoglobulin depletion from the basophil cell surface and signaling inhibition with ibrutinib. Histamine release from magnetic bead isolated platelet mitochondria was compared to platelet lysate with or without DNase treatment. Platelet lysate histamine content represented an average of <4% of total basophil histamine content. The residual histamine originating from platelet lysate was subtracted from post-reaction histamine concentrations. Atopic histories and grass, tree, and weed-specific IgE were measured in basophil donors.

Results: Robust, dose-responsive histamine release to platelet lysate was observed in two of eight healthy donors. No histamine release was observed with plasma. Reactivity did not correlate with the clinical allergic phenotype, and reactive donors were nulliparous and had no prior transfusion. Trypsin treatment of platelet lysate reduced histamine release by 39% (p=0.008). Delipidation of platelet lysate decreased histamine release by 20% (p=0.051). Dialysis, ultracentrifugation, and mild heat denaturation of platelet lysate did not significantly affect histamine release, compared to unmanipulated platelet lysate. To investigate the immunoglobulin dependence of basophil activation, in separate experiments we 1) depleted immunoglobulins, including IgE, from the basophil cell surface, and 2) inhibited immunoglobulin-mediated intracellular signaling with ibrutinib. Histamine release in response to platelet lysate significantly decreased in both cases. Finally, isolated platelet mitochondria induced minimal basophil histamine release, and DNase treatment did not inhibit activation of basophils by platelet lysate.

Conclusion: Type I immediate hypersensitivity to platelet, not plasma, proteins is a primary mechanism for ATRs. Small molecules, microparticles, and mitochondria are not significant contributors to ATRs in this human basophil model. Prior sensitization to human proteins is not required for basophil responses to platelet proteins. Donor variability and storage conditions that promote accumulation of soluble platelet-derived proteins may contribute to ATRs.