Sepsis is one of the common diseases in the ICU and the most important cause of multiple organ dysfunction syndrome in the intensive care unit. When the body is infected and the intestinal mucosal barrier function is reduced, pathogens and their metabolites can directly enter the blood, which can lead to sepsis. The pathogen of sepsis is mainly Gram-negative bacilli, and endotoxin is a component of cell wall lipopolysaccharide that is released when Gram-negative bacteria survive and die. It is an important trigger substance for systemic inflammatory response and can activate mononuclear macrophages in vivo Cells, endothelial cells, granulocytes and other inflammation-related cells, and release a variety of inflammatory mediators. The current view is that the level of plasma sepsis is closely related to the gastrointestinal dysfunction of patients and the risk of sepsis, and its level is helpful to distinguish sepsis and septic shock.
LAL test method
There are several LAL test methods, gel electrophoresis, kinetic turbidimetry, kinetic colorimetry, and endpoint colorimetry. Gel clots are not sensitive enough to be quantified. Endpoint colorimetry should not be selected because slight turbidity of the sample will affect the measured endotoxin value. Pretreated human plasma generally shows turbidity, and turbidity increases the absorbance of the reaction mixture. This can lead to false positives. For this purpose, kinetic methods are preferred because they have high sensitivity. Unspecific noise may also affect the measurement of kinetic methods.
Fig 1. Workflow.
(1) Endotoxin in serum and plasma must be pretreated
Some people try to measure endotoxins in serum and plasma by simple dilution. However, it is rarely successful, and we do not recommend it, because amidase/protease activity and serine protease inhibitors are usually present in serum and plasma. They usually cause false positives and false positives.
(2) Contamination of containers and laboratory equipment
Endotoxin and β-glucan contamination can be observed in common laboratory equipment, such as plastic sterile pipettes, pipette tips, test tubes, serum tubes, and blood collection tubes. An article should not be considered to be free of endotoxin/β-glucan if it has not been checked for endotoxin contamination. Some people use non-sterile containers to store serum/plasma samples, but these non-sterile products are often contaminated with endotoxins.
(3) Types of anticoagulants for plasma preparation
For plasma preparations, anticoagulants such as citrate, EDTA and heparin are used. Citrate and EDTA are more inhibitory than heparin. Further dilution or addition of Mg buffer may be required to overcome the inhibitory effect. If pretreatment using dilution and heating methods, heating reduces the endotoxin efficacy of the chelator in diluted plasma. In this case, you need to add magnesium or calcium before heating. The risk of heparin is the contamination of endotoxin / β-glucan because it is a natural product.
(4) Target endotoxin concentration
Because endotoxin levels in the blood are generally low, sometimes minimal dilution is required to detect lower endotoxin concentrations in a sample. Considering the pyrolysis dose of endotoxin is 5 EU / kg, some published papers report high endotoxin values in normal subjects. However, because sample noise is often considered in measurements, they are often unreliable. When reading such papers, you need to evaluate the method very carefully. Endotoxin levels in normal people may not be detected.
(5) β-glucan interference
β-glucan is often found in clinical samples, and the commercial products used may be contaminated with beta-glucan. Therefore, β-glucan blocker buffer should be used to reconstitute the LAL reagent.
(6) Noise during measurement
Unspecific turbidity changes can be observed at the beginning of the measurement. You will notice this noise when you observe the response of the measurement. This type of noise is usually observed in high immunoglobulin, high lipid, and high hemoglobin samples.
Creative BioMart offers a corresponding endotoxin testing service. You can purchase the corresponding endotoxin removal kit and related accessory products according to the needs of your own samples. We guarantee that all instruments, water, reagents, and consumables used in the experiment are free of endotoxins, and the experiment is conducted in a clean room to ensure that low levels of endotoxins are returned to the sample. In addition, we can also provide you with related services including:
|Project name||Endotoxin testing in plasma|
|Testing purpose||The causative agent of sepsis is mainly Gram-negative bacilli, and endotoxin is a component of cell wall lipopolysaccharide, which is released when gram-negative bacteria survive and die. The level of plasma sepsis is closely related to the patient's gastrointestinal dysfunction and the risk of sepsis, and its level helps to distinguish sepsis from septic shock. It is very important to detect endotoxin levels in plasma.|
|Testing cycle||3-5 days.|
|Service including||We provide you with raw data and test reports.|