Name: endotoxin activity assay for the detection of whole blood endotoxemia in critically ill patients
Uploaded: 2019-06-24T12:30:00.000+00:00
Duration: 6 min 28 s
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 consists of 5 different kinds of tubes; use each one for a different portion of the test (see section 2).
	<ol>
		<li>Use tube #1 (the &#8220;Control&#8221; tube) to measure the basal activity of the non-specific oxidative burst of patient&#8217;s neutrophils in the absence of a specific antibody.</li>
		<li>Use tube #2 (the &#8220;Sample&#8221; tube) to measure the oxidative burst in response to the LPS-antibody complex.</li>
		<li>Use tube #3 (the &#8220;Max&#8221; tube) to measure the maximal oxidative burst of patient&#8217;s neutrophils in response to an excess of endotoxin.</li>
		<li>Use tube #4 (the &#8220;LPS&#8221; tube) as a source of exogenous endotoxin.</li>
		<li>Use tube #5 (&#8220;Aliquot&#8221; tube) for blood storage. 
		NOTE: Duplicates of tubes #1, #2 and #3 are provided for a total of 8 tubes to be used for each blood sample being tested (the EA reagent bottle and quality control test can be used for all the tests contained in a pouch).</li>
	</ol>
	</li>
	<li>Collect patient blood samples in sterile tubes containing EDTA anticoagulant. Store blood samples at room temperature before running the EA test.</li>
	<li>Before starting the test, turn on the chemiluminometer and incubator shaker. Warm the incubator to the temperature of 37 &#176;C.</li>
	<li>Ideally, start processing the sample within 30 min from blood collection.</li>
</ol>
2. Endotoxin Activity Assay
<ol>
	<li>Prepare the EA test tubes for each patient&#8217;s blood sample you need to test. Put the tubes in tube racks. Then remove the caps.</li>
	<li>Using a combipipette, pipette a 1 mL volume of the EA reagent from the bottle into tubes #1 (Control tube), #2 (Sample tube) and #3 (Max tube), each one in duplicate. 
	NOTE: Pipette down the side of the tube to avoid solution splashing back up.</li>
	<li>Mix the patient blood sample by gently inverting the blood collection tube for 20 times. Then, pipette 0.5 mL of patient blood into tube #4 (LPS max tube) and tube #5 (Aliquot tube). Vortex tube #4 for 10 s.</li>
	<li>Put the tube racks with all the EA test tubes in the incubator shaker. Close the lid and incubate for 10 min at the temperature of 37&#176;C.</li>
	<li>Open the lid and remove the tube racks from the incubator shaker. Vortex tube #5 (Aliquot tube). Using a sterile tip, pipette 40 &#181;L of blood into tubes #1 and #2, in duplicate.</li>
	<li>Vortex tube #4 (LPS tube). Using the same pipette tip, pipette 40 &#181;L of blood from tube #4 into tube #3 (Max tube), in duplicate.</li>
	<li>Vortex the six final test tubes (#1, #2, #3, and respective duplicates), then place them back into their racks. 
	NOTE: Ensure that all the tubes are vortexed for the same amount of time.</li>
	<li>Put the tube racks back into the incubating shaker and close the lid. Set the incubating shaker at 100 rpm, then start the motion for 14 min.</li>
	<li>Insert the EA labeled chipcard in the chemiluminometer and press start. After the 14 min incubation, follow the instructions displayed on the chemiluminometer to read the EA tubes in the correct order.</li>
	<li>Gently vortex each tube for 10 s before placing it onto the sample holder of the chemiluminometer. Open the sample drawer and place tube #1 in the sample holder. Then, close the sample drawer and wait for the Relative Light Unit (RLU) reading.</li>
	<li>Repeat step 2.10 for tube 2 and tube 3.</li>
	<li>Repeat step 2.10 for duplicate tubes 1, 2, and 3. 
	NOTE: Try to vortex all tubes for the same amount of time during steps 2.10&#8211;2.12.</li>
	<li>After all the tubes have been processed, note that the EA results will be calculated and printed automatically. Levels are expressed as EA units and represent the mean of duplicate determinations from the same samples.</li>
	<li>Repeat steps 2.2 to 2.13 for every blood sample that needs to be tested.</li>
	<li>Once the assay has been completed, store the remaining test tubes and EA reagents at 2&#8211;8 &#176;C for up to 30 days.</li>
</ol>

<ol>
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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>

Estor SpA covered the cost of the journal&#8217;s publication and video production fee. The authors have no conflicts of interest to disclose.

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, and any healthcare provider can easily and quickly learn how to run it. The prompt identification of a high circulating EA might help in stratifying patient&#8217;s risk, triggering an earlier and more aggressive therapeutic approach. Conversely, an EA result of less than 0.40 EA units might indicate a low risk of progression to multi-organ failure17. The use of patient&#8217;s samples as their own control is simple, makes this test more sensitive and a more accurate representation of the true blood level of endotoxemia.
The use of LPS-antibody complexes and the patient&#8217;s own neutrophils protects the EA test from being possibly inhibited by other factors (e.g. plasma proteins). The same cannot be said for other test, like the LAL test, which requires the activation of a coagulation cascade. The LAL test performs well when the endotoxin is not bound by a specific receptor, but in plasma and whole blood different proteins bind LPS, interfering with the assay. Moreover, fungal products may trigger the limulus coagulation cascade, making the test less specific for GN bacteria. For these reason, the EA is superior to the still commonly adopted LAL test for the evaluation of endotoxemia18.
However, for the EA test to be reliable, it is crucial to meticulously follow the aforementioned steps. The endotoxin level of the specimen being tested is calculated by computing chemiluminescence over time, measuring basal (tube #1) and maximal (tube #3) responses for the same blood sample as reference values. Therefore, it is imperative to carefully place the three tubes in the correct order into the chemiluminometer.
The use of EA levels in clinical practice should not replace standard tests (e.g. laboratory tests and blood cultures) in the workup of a potential infectious disease. Although LPS might clearly be associated with the release of GN bacteria membrane products, elevated levels of endotoxemia have also been reported in case of infections due to other agents19. It is very well known that endotoxemia might be due to translocation of bacteria through the gut mucosa, particularly whenever tissue hypoperfusion and increased gut barrier permeability are likely to occur20,21. Under such conditions, it is possible to expect circulating LPS to be a consequence, rather than the cause, of sepsis and shock. In this scenario, the EA could provide information about the severity of the ongoing tissue injury, regardless of the bacterial etiology15. Supporting this principle, in a recent study Grimaldi et al. found the elevation of EA levels to be associated with the severity and duration of shock following out-of-hospital cardiac arrest13.
Interestingly, Virz&#237; et al. also highlighted the potential role of endotoxin (and its monitoring) in patients with type 5 cardiorenal syndrome, a condition characterized by concomitant cardiac and renal dysfunction in the setting of different systemic disorders, such as sepsis23 . Endotoxin is known to induce impairment of cardiac myocytes, although the exact pathophysiological mechanism is largely unclear. On the other hand, endotoxemia has been shown to induce renal dysfunction due to several pathways of local and systemic injury, causing impairments of renal blood flow, glomerular filtration rate, and tubular function22.
However, a few limitations of the EA must be highlighted. The influence of an ongoing antibiotic therapy on the EA result is currently not well established24. Moreover, evidence suggests that in critical ill patients a single-point early EA measurement, while useful, does not reliably predict septic shock mortality, even when greater than 0.6 units15. Serial repeated assessments might be needed, although their number and timing are currently unclear. Other authors focused on fluctuations in endotoxemia, hypothesizing that an increased daily variability might be associated with a higher degree of multi-organ dysfunction25. Lastly, an additional technical limitation to be considered is the relative scale along which the endotoxin activity is measured (0 to 1 units, with 0.01 minimum detectable increments). This makes extremely high results inevitably plateau around the maximum level, thereby making differentiations in this subgroup of patients (EA &#62; 0.9 units) harder to investigate.
In conclusion, while further studies are needed to evaluate the potential impact of monitoring endotoxin levels on clinical outcome, the EA is currently available as a quick, simple and sensitive test, which might facilitate the decision-making process of ICU clinicians in critical ill septic patients.

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><tbody><tr><td>EAA kit</td><td>Spectral Medical Inc.</td><td>EAAST-20</td><td>Package with 20 tests + 1 quality control</td></tr><tr><td>Smart Line TL</td><td>Berthold</td><td>EAASL</td><td>Luminometer</td></tr><tr><td>Incubator shaker</td><td>GRANT</td><td>ES-20</td><td>Mini-incubator shaker</td></tr><tr><td>Vortexer</td><td>VWR</td><td>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.
In the ED, he was lethargic, confused when awakened. His temperature was 39.1 &#176;C, heart rate was 125 beats per minute, blood pressure was 80/40 mmHg, respiratory rate was 20/min, and SpO2 was 94% in room air, raising to 99% with 4 L/min oxygen through a nasal cannula. Abdominal exam was normal, except for poorly-localized mild suprapubic tenderness. With difficulty, a Foley catheter was placed. A low amount of dark-colored purulent urine was drained.
A complete blood count revealed a white cell count of 18.5 x 103. Creatinine was 2.7 mg/dL, glucose was 250 mg/dL, and the lactic acid level was 4.5 mmol/L. Arterial blood gas analysis revealed mixed acidosis, with pH 7.23, pCO2 48 mmHg, pO2 88 mmHg, and HCO3- 15 mmol/L.
A central venous catheter was placed, with ultrasound guidance, into the right internal jugular vein. A blood gas analysis was obtained upon the catheter placement, revealing a 63% ScVO2 value. Aggressive fluid resuscitation was promptly started with a 30 mL/kg crystalloid bolus infusion over 30 min. Norephinephrine infusion was also initiated. The patient was transferred to the ICU with a diagnosis of septic shock, likely originating from a urinary tract infection.
In the ICU, amid microbial cultures collection and empiric antibiotic therapy administration, a whole blood sample was obtained for EA testing. The assay was rapidly performed according to the protocol presented herein.
In order to compute EA results, the chemiluminometer records: the basal luminescence of neutrophils (L1); luminescence of neutrophils activity in response to the LPS in the blood sample (L2); the maximal neutrophils activity in response to massive exposure to LPS (L3). Results are expressed as: Endotoxin Activity (EA) = (L2-L1)/(L3-L1). Therefore, the resulting EA value reflects the degree of patient&#8217;s neutrophils oxidative burst due to the presence of circulating endotoxin (L2), normalized by the highest level of luminescence which can be measured in the same blood sample in response to a supra-maximal concentration of LPS (L3). Both values are controlled for the basal luminescence of the sample (L1).
After approximately 30 min, the clinician acknowledged 0.75 EA units to be the endotoxin activity level of the patient.
An EA value 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. Results between 0.40 EA and 0.59 EA units indicates an intermediate endotoxin activity level, which represents an elevated risk for the development of severe sepsis and septic shock. Results equal or greater than 0.60 EA units indicates a high endotoxin activity level, which represents a&#160; high risk for septic shock and poor patient outcomes (Table 1).
The diagnosis of septic shock, likely due to GN bacteria, was therefore confirmed. The patient was also categorized as being at extremely high risk, in line with the evidence of concomitant organ failures and lactate level. He was furthermore treated with aggressive volume resuscitation and vasoactive support. Polymixin-B hemopurification therapy was considered, yet not implemented, due to the convincing early response of the patient to fluids and vasopressors. On day 2, Escherichia coli was confirmed as the causative agent through positive blood cultures. With the appropriate antibiotic therapy, the patient recovered, being discharged from the ICU on day 7. A second EA test was performed before ICU discharge. A result of 0.2 EA units indicated complete resolution of the biochemical cascade triggered by septic shock.
Figure 1 shows the distribution of EA values measured within 24 h in a sample population of septic shock patients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58507/58507fig1.jpg" /> 
Figure 1: Distribution of Endotoxin Activity (EA) levels measured within 24 h from septic shock onset in a population of critically ill patients (n = 107). Adapted from Bottiroli, et al. with permission15. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58507/58507fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EA units</td>
			<td>Endotoxin activity level</td>
		</tr>
		<tr>
			<td>&#60;0.40</td>
			<td>Low</td>
		</tr>
		<tr>
			<td>0.40 &#8211; 0.59</td>
			<td>Intermediate</td>
		</tr>
		<tr>
			<td>&#8805;0.60</td>
			<td>High</td>
		</tr>
	</tbody>
</table>
Table 1: Categories of Endotoxin Activity levels. EA = Endotoxin Activity.

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 ...

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9.3K Views.  Niguarda Hospital. 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.

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

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Environment

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Bioengineering

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

Immunology and Infection

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

Dosage-Dependent Induction of Endotoxemia in Pigs via LPS Infusion

Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these patients. It is caused by a dysregulated immune response to an infection. Even with optimized treatment, mortality rates remain high, which makes further insights into the pathophysiology and new treatment options necessary. Lipopolysaccharide (LPS) is a component of the cell membrane of gram-negative bacteria, which are often responsible for infections causing sepsis and septic shock. 
The severity and high mortality of sepsis and septic shock make standardized experimental studies in humans impossible. Thus, an animal model is needed for further studies. The pig is especially well suited for this purpose as it closely resembles humans in anatomy, physiology, and size. 
This protocol provides an experimental model for endotoxemic shock in pigs by LPS infusion. We were able to reliably induce changes frequently observed in septic shock patients, including hemodynamic instability, respiratory failure, and acidosis. This will allow researchers to gain valuable insight into this highly relevant condition and evaluate new therapeutic approaches in an experimental setting.

Author Spotlight: Induction of Experimental Endotoxemic Shock in Pigs for Studying Hemodynamic and Respiratory Failure

We provide a protocol for an experimental endotoxemic shock model in pigs by infusion of lipopolysaccharide.

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these ...

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

Source: Pinciroli, R. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58507">Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients.</a> J. Vis. Exp. (2019).
This video demonstrates a rapid bioassay for investigating endotoxin activity in a whole blood sample via a chemiluminescence-based method. This assay is a rapid and sensitive biomarker test for early-stage diagnosis of endotoxin-mediated sepsis.

Source: Pinciroli, R. et al. Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients. J. Vis. Exp. (2019).
This ...

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Biochemical assays

Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

Source: Neun, B. W. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58830">Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays.</a> J. Vis. Exp. (2019)
This video describes the turbidimetric format of the Limulus amoebocyte lysate, or LAL, assay to estimate endotoxin contamination in PEGylated liposomal doxorubicin nano-formulations. The turbidity of the solution following the reaction of the endotoxin with the LAL reagent helps to estimate the endotoxin in the sample.

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019)
This video ...

Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations

Source: Neun, B. W. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58830">Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays.</a> J. Vis. Exp. (2019).
This video describes a gel-clot assay to determine the presence of bacterial endotoxins in nano-formulations using Limulus Amoebocyte Lysate derived from the blood cells of horseshoe crabs. The assay helps test for endotoxin contamination of pharmaceutical products, including nano-based formulations.

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019).
This video ...

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

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Chapters in this video

Air-sampled Filter Analysis for Endotoxins and DNA Content

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations