Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Summary

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Abstract

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay's output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as “low” (<0.4 units), “intermediate” (0.4–0.59 units), or “high” (≥0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

Introduction

The Lipopolysaccharide (LPS), also known as endotoxin, is a key component of the membrane structure of Gram-negative (GN) bacteria. It makes up about 10% of the cell wall, being vital for the outer membrane integrity and homeostasis. Moreover, it is a potent activator of the host innate immune system^1,2.

In vitro exposure of innate immune system cells to LPS leads to changes in the expression of multiple genes³. Administration of very small quantities of LPS in healthy human volunteers triggers the cascade of acute systemic inflammation, whereas sepsis and se....

Protocol

The protocol is conducted according to institutional guidelines relating to the handling of human biospecimens and following the current standard operative procedures of our clinical laboratory. The use of EA data and clinical information of patients being tested follows the guidelines of our institution’s human research ethics committee.

1. Laboratory Equipment and Assay Kit Contents

1. Store the EA kit at 2–8 °C when not in use.
2. Each EA test consists of 5 different kinds of tubes; use each one for a different portion of the test (see section 2).
   1. Use tube #1 (the “Contr....

Results

A 72 year-old man was admitted to the Emergency Department (ED) of an academic urban hospital. A few days earlier he had presented to his primary care physician complaining of burning on urination. A short-course therapy with oral phosphomycin was recommended. His medical history included hypertension, uncomplicated type-2 diabetes and benign prostatic hyperplasia. His medications included enalapril, atorvastatin, tamsulosin and metformin.

Discussion

Septic shock is still nowadays associated with a mortality as high as 40%, although this rate varies according to the considered reports¹⁶. The need for novel and better biomarkers is advocated by most experts in the fields in order to aid clinicians in early diagnosis, better management, and prognostication of patients with septic shock⁶.

Performing an EA test does not require previous technical knowledge or sophisticated laboratory equipment, a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EAA kit	Spectral Medical Inc.	EAAST-20	Package with 20 tests + 1 quality control
Smart Line TL	Berthold	EAASL	Luminometer
Incubator shaker	GRANT	ES-20	Mini-incubator shaker
Vortexer	VWR	444-2790	Vortex instrument