Bench-to-bedside review: Clinical experience with the endotoxin activity assay.
Bottom Line: 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.These include ICU patients at risk of developing 'sepsis syndrome', abdominal and cardiovascular surgery patients and patients with serious traumatic injury.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.
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.
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Mentions: The endotoxin activity assay (EAA) quantifies endotoxin levels via a relative scale from 0 to 1.0 based on an internal endotoxin standard that is added to each patient sample . The assay is composed of three separate reactions all containing the patients whole blood: a blank that contains all reagents excluding anti-lipid A antibody; a sample tube that contains anti-lipid A antibody; and a maximal response tube that contains a saturating dose of exogenous endotoxin. The endotoxin acitivity is measured by stimulation of the neutrophil respiratory burst with zymosan and emission of light via reaction of oxidants (primarily HOCl) with luminol. The endotoxin activity (EA) is calculated by a simple algorithm based on light emission measured in a luminometer sensitive to light in the 450 nm range (Patient sample with antibody - Patient sample without antibody)/(Patient sample with maximal endotoxin - Patient sample without antibody). A hyperbolic dose-response relationship between endotoxin levels and EA as measured by neutrophil-dependent chemiluminescence was presented in the original methods publication . This dose-response relationship has recently been updated using a WHO referenced endotoxin standard with the current assay format (Figure 2). The dose-response relationship for WHO standardized LPS and E. coli 055:B5 LPS is depicted in Figure 3. The difference in the slope of the linearized plots is due to a higher per unit mass content of lipid A in the WHO standardized LPS (200 pg/EU) compared to the E. coli 055:B5 LPS (1,200 pg/EU). The most sensitive part of the dose-response curve (largest change in EAA per LPS dose) was empirically designed to cover endotoxin concentrations that were found in patients with confirmed Gram-negative infection but absence of septic shock (0.1 to 0.3 EU/ml; unpublished results). The precision of the assay using an automated chemiluminometer (LB 953, Berthold) yielded a CV (co-efficient of variation, standard deviation divided by mean times 100) of less than 10% at EAs above 0.4 (US FDA submission, MEDIC trial). Subsequent studies using a manual luminometer (Berthold Smartline) by Wahl and colleagues  have documented CVs between 11.9% and 18.6% for EA values between 0.38 and 0.7 (suggested normal low <0.4 EA units, intermediate level ≥0.40 and <0.6, high level ≥0.6 EA units). This group also independently validated the normal reference range using a healthy population of 43 men and 57 women aged 32 ± 14 years as <0.42 EA units (95% cutoff value). After extensive review of EAA results in a total of 469 patients, the US FDA determined that steroids for septic shock, despite inhibiting neutrophil function, had no influence on EAA results (FDA review EAA assay). Unlike LAL-based assays, the EAA is insensitive to extrinsic environmental endotoxin contamination, as demonstrated in Figure 4. In the absence of blood proteins the assay is approximately 500 to 1,000 times less sensitive to endotoxin due to the necessity for blood proteins to disaggregate and expose the cryptic lipid A domain in whole blood for efficient binding to the anti-lipid A antibody. Even at endotoxin concentrations of 1,000 pg (1.3 EU, E. coli 055:B5 LPS) in the assay buffer the EA is not elevated above the suggested normal cutoff of 0.4 EA units (Figure 4). Assay specificity for Gram-negative bacteria possessing a lipid A epitope was established with a lack of reactivity against Gram-positive and fungal antigens . Assay specificity was also confirmed by inhibition studies with the lipid A binding peptide polymyxin B in ICU patient samples  (Figure 5).