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

Abstract

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. The therapeutic use of intra-articular A2M requires a standardized protocol for its preparation, coupled with 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, resulting in the production of 12 mL of A2MRP. The concentrations of A2MRP and certain cytokines were determined by enzyme-linked immunosorbent assay 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. insulin-like growth factor 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 osteoarthritis.