Off-the-Shelf Cryopreserved Platelets to De-Risk Intravenous Immunoglobulin G (IVIg) Lots That Cause Platelet Activation

Introduction

Intravenous Immunoglobulin G (IVIg) is a pooled antibody Immunoglobulin G product that is used for a large and ever-growing number of medical indications. It is also known to be associated with an increased risk of thromboembolic complications such that the Food and drug administration (FDA) has mandated a “black box” warning alerting physicians to this risk. Particular lots of IVIg preparations have been recalled due to a higher-than-expected rate of thromboembolic adverse events (“Evaluation of the Risk of Thromboembolic Events After Immunoglobulin Administration, 2006-2012”. Sentinel Product Assessment, US FDA, 7/13/2017). While a number of mechanisms have been proposed to explain the thromboembolic risk, platelet activation induced by small amounts of aggregated IgGs in IVIg preparations remains the leading hypothesis. Currently, testing of IVIg lots specifically looking at their ability to induce platelet activation is not routinely performed due to challenges with acquisition of fresh platelets for testing, and lack of availability of technically simple platelet activation endpoints. Recent breakthroughs in platelet storage technology have demonstrated that platelet viability can be preserved for long periods (months-years) by controlled-rate cryopreservation which also allows platelets to retain FcγRIIa-mediated signaling (FcγRIIa is the IgG receptor on platelets). This technology has been successfully leveraged for the detection of platelet-activating heparin-induced thrombocytopenia (HIT) and Vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies (Blood, 140 (25) 2722-2729, 2022).

Goals of the study

We hypothesized that long-term stored cryopreserved platelets could be used to detect platelet-activating IVIg preparations using a technically simple ELISA endpoint that measures levels of thrombospondin-1 (TSP-1), a platelet granule protein, released upon platelet activation. If successful, this tool could help de-risk IVIg from the standpoint of thrombotic adverse reactions.

Methods

Aggregated IgG was prepared by heat treatment of IVIg (Pel-Freez- Rogers, AR) as previously described (Immunol. Lett. (15) 311-316, 1987). Platelets were cryopreserved as previously described (Blood, 140 (25) 2722-2729, 2022) with minor modifications. Cryopreserved platelets were thawed and treated with heat-aggregated or untreated IVIg at concentrations ranging from 19 to 75mcg/mL. After incubation, supernatants were collected by centrifugation and were assayed for TSP-1 by an antigen capture ELISA which has been shown previously to correlate with platelet activation.

Results

10-month-old “off-the-shelf” cryopreserved platelets treated with buffer control displayed minimal platelet activation as measured by the TSP1 ELISA (Optical density, OD: 0.21). Similarly, platelets treated with IVIg (non-aggregated IgG) at concentrations ranging from 19 to 75mcg/mL displayed little-to-no platelet activation in the TSP1 ELISA (OD: 0.22-0.29). In contrast, cryopreserved platelets treated with heat aggregated IVIg demonstrated marked platelet activation in the TSP1 ELISA with ODs in the 0.60-0.82 range, correlating with increasing concentrations of heat-aggregated IVIg in the reaction.

Conclusions

De-risking IVIg from a prothrombotic standpoint has been challenging due to the need for fresh platelets for testing and the lack of technically simple means to assess platelet activation. Here, we leveraged breakthrough platelet storage technology coupled to a technically simple ELISA endpoint that allowed for rapid and easy identification of platelet activating forms of IVIg. This easy-to-use self-contained test kit could be implemented as a quality control/patient safety tool to de-risk IVIg preparations from the standpoint of platelet activation-mediated thrombosis. Future studies aim to test a large number of IVIg lots, including “problem” lots associated with thromboembolic events to assess the incidence of platelet activating IgG molecules in IVIg preparations.

Acknowledgments

This work was supported in part by a grant from the National Institutes of Health (C.G.J.: HL147734).