title: Is Endotoxemia in Stable Hemodialysis Patients an Artefact? Limitations of the Limulus Amebocyte Lysate Assay and Role of (1→3)-β-D Glucan.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Lister Renal Unit, Hertfordshire, United Kingdom.|University of Hertfordshire, Hertfordshire, United Kingdom.
authors.forename: Jonathan
authors.identifier: http://orcid.org/0000-0002-1811-9806
authors.initials: J
authors.lastname: Wong
authors.affiliation: Associates of Cape Cod Inc., East Falmouth, Massachusetts, United States of America.
authors.forename: Yonglong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: University of Hertfordshire, Hertfordshire, United Kingdom.
authors.forename: Ashish
authors.initials: A
authors.lastname: Patidar
authors.affiliation: Lister Renal Unit, Hertfordshire, United Kingdom.|University of Hertfordshire, Hertfordshire, United Kingdom.
authors.forename: Enric
authors.initials: E
authors.lastname: Vilar
authors.affiliation: Associates of Cape Cod Inc., East Falmouth, Massachusetts, United States of America.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Finkelman
authors.affiliation: Lister Renal Unit, Hertfordshire, United Kingdom.|University of Hertfordshire, Hertfordshire, United Kingdom.
authors.forename: Ken
authors.initials: K
authors.lastname: Farrington
mesh_terms: D000368:Aged; D016477:Artifacts; D002021:Buffers; D003430:Cross-Sectional Studies; D019446:Endotoxemia / Q000097:blood* / Q000175:diagnosis / Q000209:etiology; D004731:Endotoxins / Q000097:blood; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D008033:Limulus Test / Q000379:methods*; D008297:Male; D008875:Middle Aged; D006435:Renal Dialysis / Q000009:adverse effects; D047071:beta-Glucans / Q000097:blood*
chemical_list: D002021:Buffers; D004731:Endotoxins; D047071:beta-Glucans