Name: endotoxin activity assay for the detection of whole blood endotoxemia in critically ill patients
Uploaded: 2019-06-24T12:30:00
Description: Watch this Scientific Journal Video about Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients at JoVE.com

We thank Paolo Bragan&#242; and Lisa Mathiasen, Ph.D. for their review of the assay protocol methodology. Dario Winterton, MD provided substantial help reviewing the manuscript for English language proficiency.

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&#8217;s human research ethics committee.
1. Laboratory Equipment and Assay Kit Contents
<ol>
	<li>Store the EA kit at 2&#8211;8 &#176;C when not in use.</li>
	<li>Each EA test consis...

<ol>
	<li>Akira, S., Takeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signalling.">Toll-like receptor signalling.</a> Nature Reviews Immunology. 4 (7), 499-511 (2004).</li><li>Takeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+integration+of+innate+immune+recognition+systems:+the+Toll-like+receptors.">Evolution and integration of innate immune recognition systems: the Toll-like receptors.</a> Journal of Endotoxin Research. 11 (1), 51-55 (2005).</li><li>Ulevitch, R. J., Tobias, P. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardiovascular+response+of+normal+humans+to+the+administration+of+endotoxin.">The cardiovascular response of normal humans to the administration of endotoxin.</a> The New England Journal of Medicine. 321 (5), 280-287 (1989).</li><li>Singer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Third+International+Consensus+Definitions+for+Sepsis+and+Septic+Shock+(Sepsis-3).">The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).</a> JAMA: The Journal of the American Medical Association. 315 (8), 801-810 (2016).</li><li>Gupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture-Negative+Severe+Sepsis:+Nationwide+Trends+and+Outcomes.">Culture-Negative Severe Sepsis: Nationwide Trends and Outcomes.</a> Chest. 150 (6), 1251-1259 (2016).</li><li>Ikeda, T., Ikeda, K., Suda, S., Ueno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+the+endotoxin+activity+assay+as+a+biomarker+to+assess+the+severity+of+endotoxemia+in+critically+ill+patients.">Usefulness of the endotoxin activity assay as a biomarker to assess the severity of endotoxemia in critically ill patients.</a> Innate Immunity. 20 (8), 881-887 (2014).</li><li>Dellinger, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Targeted+Polymyxin+B+Hemoperfusion+on+28-Day+Mortality+in+Patients+With+Septic+Shock+and+Elevated+Endotoxin+Level.">Effect of Targeted Polymyxin B Hemoperfusion on 28-Day Mortality in Patients With Septic Shock and Elevated Endotoxin Level.</a> The EUPHRATES Randomized Clinical Trial. JAMA: The Journal of the American Medical Association. 320 (14), 1455-1463 (2018).</li><li>Marshall, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+prognostic+implications+of+endotoxemia+in+critical+illness:+results+of+the+MEDIC+study.">Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC study.</a> The Journal of Infectious Diseases. 190 (3), 527-534 (2004).</li><li>Levin, J., Bang, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clottable+protein+in+Limulus;+its+localization+and+kinetics+of+its+coagulation+by+endotoxin.">Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin.</a> Thrombosis et Diathesis Haemorrhagica. 19 (1), 186-197 (1968).</li><li>Marshall, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+endotoxin+activity+in+critically+ill+patients+using+whole+blood+neutrophil+dependent+chemiluminescence.">Measurement of endotoxin activity in critically ill patients using whole blood neutrophil dependent chemiluminescence.</a> Critical Care. 6 (4), 342-348 (2002).</li><li>Grimaldi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Level+of+Endotoxemia+Following+Out-of-Hospital+Cardiac+Arrest+Is+Associated+With+Severity+and+Duration+of+Postcardiac+Arrest+Shock.">High Level of Endotoxemia Following Out-of-Hospital Cardiac Arrest Is Associated With Severity and Duration of Postcardiac Arrest Shock.</a> Critical Care Medicine. 43 (12), 2597-2604 (2015).</li><li>Klein, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EUPHRATES+trial+(Evaluating+the+Use+of+Polymyxin+B+Hemoperfusion+in+a+Randomized+controlled+trial+of+Adults+Treated+for+Endotoxemia+and+Septic+shock):+study+protocol+for+a+randomized+controlled+trial.">The EUPHRATES trial (Evaluating the Use of Polymyxin B Hemoperfusion in a Randomized controlled trial of Adults Treated for Endotoxemia and Septic shock): study protocol for a randomized controlled trial.</a> Trials. 15, 218 (2014).</li><li>Bottiroli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+clinical+significance+of+early+high+Endotoxin+Activity+in+septic+shock:+An+observational+study.">Prevalence and clinical significance of early high Endotoxin Activity in septic shock: An observational study.</a> Journal of Critical Care. 41, 124-129 (2017).</li><li>Kaukonen, K. M., Bailey, M., Suzuki, S., Pilcher, D., Bellomo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+related+to+severe+sepsis+and+septic+shock+among+critically+ill+patients+in+Australia+and+New+Zealand.">Mortality related to severe sepsis and septic shock among critically ill patients in Australia and New Zealand.</a> JAMA: The Journal of the American Medical Association. 311 (13), 1308-1316 (2014).</li><li>Biagioni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+activity+levels+as+a+prediction+tool+for+risk+of+deterioration+in+patients+with+sepsis+not+admitted+to+the+intensive+care+unit:+a+pilot+observational+study.">Endotoxin activity levels as a prediction tool for risk of deterioration in patients with sepsis not admitted to the intensive care unit: a pilot observational study.</a> Journal of Critical Care. 28 (5), 612-617 (2013).</li><li>Roth, R. I., Levin, F. C., Levin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+detection+of+bacterial+endotoxin+in+plasma+with+the+Limulus+test.">Optimization of detection of bacterial endotoxin in plasma with the Limulus test.</a> The Journal of Laboratory and Clinical Medicine. 116 (2), 153-161 (1990).</li><li>Yaguchi, A., Yuzawa, J., Klein, D. J., Takeda, M., Harada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+intermediate+levels+of+the+Endotoxin+Activity+Assay+(EA)+with+other+biomarkers+in+the+assessment+of+patients+with+sepsis:+results+of+an+observational+study.">Combining intermediate levels of the Endotoxin Activity Assay (EA) with other biomarkers in the assessment of patients with sepsis: results of an observational study.</a> Critical Care. 16 (3), (2012).</li><li>Earley, Z. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+Injury+Alters+the+Intestinal+Microbiome+and+Increases+Gut+Permeability+and+Bacterial+Translocation.">Burn Injury Alters the Intestinal Microbiome and Increases Gut Permeability and Bacterial Translocation.</a> PLoS One. 10 (7), (2015).</li><li>Munster, A. M., Smith-Meek, M., Dickerson, C., Translocation Winchurch, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidental+phenomenon+or+true+pathology?.">Incidental phenomenon or true pathology?.</a> Annals of Surgery. 218 (3), 321 (1993).</li><li>Clementi, A., Virz&#236;, G. M., Brocca, A., Ronco, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Endotoxin+in+the+Setting+of+Cardiorenal+Syndrome+Type+5.">The Role of Endotoxin in the Setting of Cardiorenal Syndrome Type 5.</a> Cardiorenal Medicine. 7 (4), 276-283 (2017).</li><li>Virz&#236;, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiorenal+Syndrome+Type+5+in+Sepsis:+Role+of+Endotoxin.">Cardiorenal Syndrome Type 5 in Sepsis: Role of Endotoxin.</a> in Cell Death Pathways and Inflammation. Kidney and Blood Pressure Research. 41 (6), 1008-1015 (2016).</li><li>Mignon, F., Piagnerelli, M., Van Nuffelen, M., Vincent, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+empiric+antibiotic+treatment+on+plasma+endotoxin+activity+in+septic+patients.">Effect of empiric antibiotic treatment on plasma endotoxin activity in septic patients.</a> Infection. 42 (3), 521-528 (2014).</li><li>Klein, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+variation+in+endotoxin+levels+is+associated+with+increased+organ+failure+in+critically+ill+patients.">Daily variation in endotoxin levels is associated with increased organ failure in critically ill patients.</a> Shock. 28 (5), 524-529 (2007).</li></ol>

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...

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 system1,2.
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 septic shock may arise with higher endotoxin concentrations4,5.
Sepsis is a life-threatening condition which, if not promptly recognized, can lead to multi-organ failure and death. Septic patients must be treated in a timely manner, with aggressive resuscitation, adequate antibiotic therapy, optimal source control, and prompt organ support strategies. The diagnosis of the etiology of sepsis is primarily based on clinical recognition and culture-based pathogen detection6. However, results of microbial cultures may take up to 48 h and are inconclusive in up to 30% of cases7. Early identification and intervention may lead to better patient outcomes. In patients in whom sepsis is suspected, decisions are often made on the basis of physiological and biochemical parameters, without a clear sign of endotoxemia.
The measurement of the Endotoxin Activity (EA) can be obtained by means of a commercial assay (see Table of Materials) in whole blood. It can be used as a biomarker of systemic endotoxemia for the early stratification of disease severity, particularly in patients at risk for developing septic shock8. The assay was used to guide Polymyxin B hemoperfusion therapy in a recently published double-blind randomized-controlled clinical trial in patients with septic shock9. In critically ill patients, the MEDIC study showed increased EA levels to be associated with multiple organ dysfunction, intensive care unit (ICU) length of stay, and mortality10.
Different assays have been developed to detect endotoxin. The Limulus Amoebocyte Lysate (LAL) assay, either as a gel-clot, turbidimetric, or chromogenic test, has been so far the most frequently adopted for the estimation of serum endotoxin. It is based on the ability of endotoxin to induce coagulation of the hemolymph of the horseshoe crab, Limulus polyphemus. However, this assay has some limitations in terms of specificity. In particular, it can also be activated by microbial products other than endotoxin, such as components of the fungal cell wall, and it can be inhibited by various human plasma proteins11.
During the last decade the measurement of EA has been developed and validated as a biomarker of circulating endotoxemia. Compared to the LAL test, EA is quicker and easier to implement in the clinical setting. Moreover, it has been shown to be more accurate than LAL in whole blood, with increased sensitivity and specificity, both in vitro and in vivo12.
Despite its initial implementation as an early diagnostic tool for the rapid identification of GN bacteria as sepsis causative agents, the EA level has also been studied as a biomarker of disease severity. In this context, it has been shown to be particularly useful to assess the hypoperfusion state due to ongoing critical illness, such as septic shock or post-cardiac arrest syndrome13. More recently, since the development of hemopurification systems, a positive EA result has also been proposed as a screening tool to accurately identify potential candidates for such therapy14. We recently conducted an observational retrospective study on the prevalence and clinical significance of early high levels of EA in 107 patients with septic shock. In line with other recent results, we found that EA is a promising marker of disease severity in patients with septic shock15.
The aim of the present manuscript is to describe the method to perform the EA assay, either at the bedside or in the laboratory, and to describe its potential use in a representative scenario of septic shock. This technique can detect LPS activity by measuring the enhanced oxidative burst in neutrophils following their priming by complexes of an anti-endotoxin antibody and LPS. The increased respiratory burst is detected by a chemiluminometer and the amount of light emitted is considered proportional to the amount of endotoxin in the blood sample. The assay requires few reagents, takes about 30 min to perform and uses as little as 40 &#181;L of whole blood12.

<table border="0" cellpadding="0" cellspacing="0" style="width:727px;" width="726">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">EAA kit</td>
			<td style="width:141px;">Spectral Medical Inc.</td>
			<td style="width:133px;">EAAST-20</td>
			<td style="width:268px;">Package with 20 tests + 1 quality control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Smart Line TL</td>
			<td style="width:141px;">Berthold</td>
			<td style="width:133px;">EAASL</td>
			<td>Luminometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Incubator shaker</td>
			<td style="width:141px;">GRANT</td>
			<td style="width:133px;">ES-20</td>
			<td>Mini-incubator shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Vortexer</td>
			<td style="width:141px;">VWR</td>
			<td style="width:133px;">444-2790</td>
			<td>Vortex instrument</td>
		</tr>
	</tbody>
</table>

endotoxin activity assay for the detection of whole blood endotoxemia in critically ill patients

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&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

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.
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Watch this Scientific Journal Video about Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients at JoVE.com

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

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.

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

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.

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