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

Sepsis can lead to multi-organ failure and death. The endotoxin is a fundamental component of gram-negative bacteria. Elevated circulating levels of endotoxin might identify patient at higher risk to develop a more severe disease.

The Endotoxin Activity Assay can be used as a simple, quick, and reliable biomarker of systemic endotoxemia. The early detection of this toxin might help clinicians treating patients with septic shock. In patient with septic shock, the early identification of a rapidly evolving condition might prompt a quicker and more intensive life support therapy, potentially leading to better outcomes.

Demonstrating this procedure will be Lisa Mathiasen, a molecular biologist who collaborates with our research group. To begin this procedure, set out an EA test kit and unpack the five different kinds of tubes. Use tube number one, the Control tube, to measure the basal activity of the non-specific oxidative burst of patient's neutrophils in the absence of a specific antibody.

Use tube number two, the Sample tube, to measure the oxidative burst in response to the LPS-antibody complex. Use tube number three, the Max tube, to measure the maximal oxidative burst of patient's neutrophils in response to an excess of endotoxin. Use tube number four, the LPS tube, as a source of exogenous endotoxin.

And use tube number five, the Aliquot tube, for blood storage. Collect patient blood samples in sterile tubes containing EDTA anticoagulant, and store them at room temperature before running the EA test. Before starting the test, turn on the chemiluminometer and incubator shaker and warm the incubator to 37 degrees Celsius.

First, place the EA tubes to be used in tube racks, and remove the caps. Use a combipipette to transfer one milliliter of the EA reagent from the bottle into tubes number one, number two, and number three by pipetting down the side of the tubes. Gently invert the blood collection tube 20 times to mix the patient's blood sample.

Then, pipette 0.5 milliliters of patient blood into tube number four and tube number five. Vortex tube number four for 10 seconds. Place the tube racks with all of the EA test tubes into the incubator shaker.

Close the lid, and incubate at 37 degrees Celsius for 10 minutes. After this, open the incubator lid and remove the tube racks from the shaker. Vortex tube number five.

Using a sterile tip, pipette 40 microliters of blood into tubes number one and number two, in duplicate. Vortex tube number four. Using the same pipette tip, transfer 40 microliters of blood from tube number four into tube number three, in duplicate.

Vortex the six final test tubes one by one. Place the tube racks back into the incubating shaker, and close the lid. Set the shaker to 100 rpm, and start the motion for 14 minutes.

Insert the EA labeled chipcard into the chemiluminometer, and press Start. After the 14-minute incubation, follow the instructions displayed on the chemiluminometer. Gently vortex each tube for 10 seconds before placing it onto the sample holder of the chemiluminometer.

Open the sample drawer, and place tube number one into the sample holder. Then, close the sample drawer, and wait for the relative light unit reading. Repeat this reading process for tubes two and three and all duplicates, as outlined in the text protocol.

After all of the tubes have been processed, the EA results will be calculated and printed automatically. Repeat the entire assay process for each blood sample that needs to be tested. Store the EA Assay kit and its components in the original sealed package at a temperature between two and 25 degrees Celsius.

In this study, an Endotoxin Activity Assay is performed to detect whole blood endotoxemia in critically ill patients. An EA value of less than 0.40 EA units indicates a low endotoxin activity level, equal to a low circulating LPS concentration, which represents a low risk of progression to a severe disease state. A result between 0.40 and 0.59 EA units indicates an intermediate endotoxin activity level, which represents an intermediate risk for the development of severe sepsis and septic shock.

Results equal to or greater than 0.60 EA units indicates a high endotoxin activity level, which represents a high risk for septic shock and poor patient outcomes. Blood from tubes number four and five must be mixed with the correct test tube. Extra care must be taken to follow the correct order indicated in the protocol.

Every tube is run in duplicates, potentially leading to mistakes in this phase. After its introduction, the EA Assay has been proposed as a biomarker for the risk stratification of patient with systemic hypoperfusion. Also, it has been studied as a screening test to select patients for advanced hemopurification treatment strategies.