Preparation of alpha-2 macroglobulin–rich plasma from human blood—technique and compositional analysis of plasma, platelet-rich plasma, platelet-poor plasma, and alpha-2 macroglobulin–rich plasma

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Introduction Alpha-2 macroglobulin (A2M) has been identified as an inhibitor of matrix metalloproteinases, interleukin-1β and other factors that contribute to post-traumatic osteoarthritis (OA). The therapeutic use of intra-articular A2M requires a standardized protocol for its preparation, coupled to compositional analysis of the product. Objectives 1. To develop and describe a protocol for producing A2M-rich plasma (A2MRP) from platelet-poor plasma (PPP), which is a byproduct of platelet-rich plasma (PRP) production. 2. To provide a comprehensive analysis of A2M and cytokine levels in plasma, PRP, PPP, and A2MRP. Methods Ten patients undergoing standard preparation of PRP with the Arthrex Angel® system consented to have their PRP and by-products analyzed. Sixty mL of PPP were filtered through a Hemocor Junior High Performance Hemoconcentrator (HPH Junior), resulting in production of 12 mL of A2MRP. The concentrations of A2MRP and certain cytokines were determined by ELISA and multiplexing. A2M bioactivity was confirmed by trypsin inhibition. Results Mean A2M concentrations in plasma, PRP, PPP and A2MRP were 1.56, 1.62, 1.62, and 6.66 mg/mL, respectively. IGF-1 concentrations were measured as 68.82, 51.35, 69.23, and 257.63 ng/mL in plasma, PRP, PPP and A2MRP respectively. Multiplex analysis showed alterations in the concentrations of several cytokines and growth factors in PRP compared to A2MRP, plasma and PPP. The A2M within A2MRP was biologically active. Conclusions Our results demonstrated that ultrafiltration of PPP is a reliable, quantitative method for concentrating active A2M. The levels of certain cytokines and growth factors were also affected. These data pave the way for clinical trials of intra-articular A2MRP in OA.