Is Endotoxemia in Stable Hemodialysis Patients an Artefact? Limitations of the Limulus Amebocyte Lysate Assay and Role of (1→3)-β-D Glucan.

BACKGROUND: Elevated blood endotoxin levels are frequently reported in the dialysis population and are strongly linked with inflammation, a major predictor of mortality. Virtually all studies have employed the Limulus Amoebocyte Lysate (LAL) assay to detect endotoxin. However this assay is not endotoxin-specific and can be activated by (1→3)-β-glucan (BG), a component of fungal cell walls leading to false positive signals. Very few studies have taken account of this. We examined the influence of BG-based activation of the LAL assay on the detection of endotoxemia in this setting. METHOD: We measured plasma endotoxin levels in 50 hemodialysis patients with and without the use of BG-blocking buffers. These buffers inhibit BG activation of the LAL assay to ensure that any signal detected is endotoxin-specific. Blood samples were measured for BG, interleukin-6 (IL-6), tumor necrosis factor-alfa (TNF-α) to examine the association between endotoxin signals, BG and inflammation. RESULTS: Endotoxin signals were detected in 50% of patients. On repeat measurement with a BG-blocking buffer, all detected endotoxin signals were extinguished. No patient had detectable endotoxemia. Plasma BG levels were significantly elevated in 58% of patients and were higher in those with detectable endotoxin signals using the LAL assay without BG-blocking buffers (78vs.54pg/mL;p<0.001). Endotoxin signal and BG levels did not correlate with levels of TNF-α or IL-6. CONCLUSION: Use of the LAL assay for blood endotoxin detection in dialysis patients has its limitations due to high blood BG. Endotoxemia frequently reported in non-infected hemodialysis patients may be artefactual due to BG interference.