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 septic shock may arise with higher endotoxin concentrations^4,5.

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 µL of whole blood¹².

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 “Control” tube) to measure the basal activity of the non-specific oxidative burst of patient’s neutrophils in the absence of a specific antibody.
   2. Use tube #2 (the “Sample” tube) to measure the oxidative burst in response to the LPS-antibody complex.
   3. Use tube #3 (the “Max” tube) to measure the maximal oxidative burst of patient’s neutrophils in response to an excess of endotoxin.
   4. Use tube #4 (the “LPS” tube) as a source of exogenous endotoxin.
   5. Use tube #5 (“Aliquot” 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).
3. Collect patient blood samples in sterile tubes containing EDTA anticoagulant. Store blood samples at room temperature before running the EA test.
4. Before starting the test, turn on the chemiluminometer and incubator shaker. Warm the incubator to the temperature of 37 °C.
5. Ideally, start processing the sample within 30 min from blood collection.

2. Endotoxin Activity Assay

1. Prepare the EA test tubes for each patient’s blood sample you need to test. Put the tubes in tube racks. Then remove the caps.
2. 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.
3. 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.
4. 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°C.
5. Open the lid and remove the tube racks from the incubator shaker. Vortex tube #5 (Aliquot tube). Using a sterile tip, pipette 40 µL of blood into tubes #1 and #2, in duplicate.
6. Vortex tube #4 (LPS tube). Using the same pipette tip, pipette 40 µL of blood from tube #4 into tube #3 (Max tube), in duplicate.
7. 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.
8. 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.
9. 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.
10. 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.
11. Repeat step 2.10 for tube 2 and tube 3.
12. 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 2.10–2.12.
13. 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.
14. Repeat steps 2.2 to 2.13 for every blood sample that needs to be tested.
15. Once the assay has been completed, store the remaining test tubes and EA reagents at 2–8 °C for up to 30 days.

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