Bench-to-bedside review: Clinical experience with the endotoxin activity assay

Abstract

Endotoxin detection in human patients has been a difficult challenge, in part due to the fact that the conserved active portion of the molecule (lipid A) is a relatively small epitope only amenable to binding by a single ligand at any one instance and low levels (pg/ml) are capable of stimulating the immune system. The endotoxin activity assay, a bioassay based on neutrophil activation by complement opsonized immune complexes of lipopolysaccharide (LPS), has allowed the specific detection of the lipid A epitope of LPS in a rapid whole blood assay format. This review summarizes diagnostic studies utilizing the endotoxin activity assay in a variety of hospital patient populations in whom endotoxin is postulated to play a significant role in disease etiology. These include ICU patients at risk of developing 'sepsis syndrome', abdominal and cardiovascular surgery patients and patients with serious traumatic injury. Significant features of these studies include the high negative predictive value of the assay (98.6%) for rule out of Gram-negative infection, ability to risk stratify patients progressing to severe sepsis (odds ratio 3.0) and evidence of LPS release in patients with gut hypoperfusion. Preliminary studies have successfully combined the assay with anti-LPS removal strategies to prospectively identify patients who might benefit from this therapy with early evidence of clinical benefit.

Figure 1

Reaction mechanism for endotoxin activity assay utilizing anti-lipopolysaccharide monoclonal antibody and autologous whole blood. Complement opsonised IgM-lipopolysaccharide (LPS) complexes interact with CR1 and CR3 complement receptors on neutrophils, resulting in a priming of the NADPH oxidase complex. The neutrophil respiratory burst is evoked using zymosan and measured by chemiluminescent emission from luminol. The magnitude of respiratory burst is proportional to the logarithmic concentration of LPS in the sample.

Figure 2

Typical patient dose response to endotoxin. Two response curves are illustrated utilizing the second International (WHO) lipopolysaccharide (LPS) standard preparation (E. coli 0113:H10:K, 10 EU/ng) and LPS from E. coli 055:B5 (3.58 EU/ng) currently used in the endotoxin activity (EA) assay.

Figure 3

Linearized dose response with endotoxin acitivity plotted against the log(10) lipopolysaccharide concentration. The difference in reactivity between the two lipopolysaccharide (LPS) preparations relates to differences in purity, polysaccharide chain length and lipid A structure. EAA, endotoxin activity assay.

Figure 4

Assay contamination study. Endotoxin activity (EA) assay buffer (1 ml) was spiked with either 230 pg (n = 3) or 1,000 pg (n = 3) of E. coli 055:B5 lipopolysaccharide (LPS) (3.58 EU/ng). Different control patients were used for each dose of LPS. Average EAA values are depicted in blue, error of measurement (1 standard deviation from mean) depicted in red At 1,000 pg per assay the endotoxin activity was statistically higher than the control (*P < 0.05, paired Student t-test) but still below the normal cutoff of 0.4.

Figure 5

Inhibition study with polymyxin B. Fifteen consecutive ICU patients with elevated endotoxin levels had endotoxin acitivity assays repeated following addition of polymyxin B to their blood (300 μg/ml for 20 minutes at 37°C). The dose response for the second International WHO standard E. coli 0113:H10:K lipopolysaccharide (LPS) was used to estimate the endotoxin concentration in the patient blood samples.

Figure 6

Endotoxin activity assay scattergram values for patients (n = 42) undergoing polymyxin B column hemoperfusion. Mean endotoxin activity assay (EAA) values are indicated in bold. Samples were drawn before perfusion (baseline, n = 42) and at 4 (n = 42) and 12 hours post-column treatment (n = 42). At 4 hours the mean EAA was 0.57 (*P < 0.05 versus baseline) and 0.45 at 12 hours (**P < 0.01 versus baseline). All patients showed a decline in EAA values at 12 hours post-column treatment. There was no placebo control group in this study. The red circles highlight the significant drop in mean EAA values from pre-treatment to 12 hours post-treatment with an estimated 50-100 fold reduction in endotoxin concentration.