Leukoreduction system chambers provide a valuable source of functional monocytes for the monocyte activation test by comparison with internationally validated methods

Abstract

Despite being added to the European Pharmacopoeia in 2010 and strongly supported by the European directive enforcing the "3R's" - Replace, Reduce and Refine, uptake of the monocyte activation test (MAT) in preference over the rabbit pyrogen test for the detection of pyrogens has been limited. This has been attributed to the difficulty in sourcing human monocytes due to the necessity of phlebotomy. This study has attempted to address this issue by evaluating cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from leukoreduction system chambers (LRSCs), a readily available by-product of platelet apheresis, as a source of monocytes for the MAT. Validation was performed by direct comparison with the two most commonly employed primary monocyte sources: fresh whole blood (WB) and PBMCs from fresh blood, assessing their ability to detect a panel of toll-like receptor (TLR) ligands including Pam3CSK4, Lipoteichoic acid, Peptidoglycan, Poly(I:C) and Flagellin, as well as two different endotoxin sources, with IL-1β and IL-6 as the readouts. All three cell sources were able to detect the pyrogens included in the study with comparable sensitivities, with the exception of TLR3 ligand Poly(I:C). The WB assay produced quantifiable, but significantly lower cytokine levels with every pyrogen tested than either of the PBMCs sources used. LRSCs provided an ample and convenient source of PBMCs which were successfully cryopreserved, providing cell banks for each donor, shown to maintain stability for at least a year. The use of cryopreserved PBMCs reduced the time and effort required to set up an assay, and the availability of single donor cell banks will allow investigations into assay variables in the absence of inter-donor variability. Significantly higher sensitivity to Pam3CSK4 was observed with a proportion of donors. This was found to correlate to single nucleotide polymorphisms rs4833095 and rs5743618 of TLR1. This evidence, along with the wide range of other SNPs identified in TLR regions without known biological function, supports caution in the practice of pooling donor cells in order to overcome donor-to-donor variation.

Keywords: Endotoxin; MAT; Monocyte activation test; Pam(3)CSK(4); Pyrogen; TLR.