Endotoxemia is usually reported in patients undergoing hemodialysis and is associated with the pathogenesis of systemic inflammation. A deformed cell lysate (LAL) assay is the most commonly used blood endotoxin testing assay. This assay has two kinetic changes: turbidimetry and chromometry, and it is unclear which assay is better at detecting endotoxins in uremia patients. Choosing the best LAL technology for endotoxin testing in hemodialysis patients is important to better understand the sequelae of endotoxemia and develop endotoxin reduction strategies for this population. Kinetic turbidimetry has higher accuracy and precision than chromophoresis and is the best LAL technique for endotoxin testing in hemodialysis patients, although these findings should be validated using LAL reagents from other sources.
The Chromogenic LAL Assay is an improvement of the LAL/TAL method using a chromogenic substrate to detect the presence of endotoxin in solution. The LAL assay is the most commonly used endotoxin testing assay. LAL is derived from extracts of primitive amoebocytes present in the haemolymph of the horseshoe crab. The lipid A component of endotoxin interacts with pro-clotting enzymes present in LAL activating a coagulation cascade resulting in gelation and eventual clot formation . High endotoxin content samples lead to rapid gelation. The rate of increase in turbidity can be measured using a spectrophotometer to quantify the endotoxin content of a sample. This technique is known as the turbidimetric LAL assay. A variation of this technique involves the addition of chromogenic substrates to LAL which undergoes a colour change in the presence of endotoxin. The chromogenic LAL assay has been the most commonly used endotoxin testing method in previous human studies and dialysis literature. However, in Japan, the turbidimetric LAL assay is routinely used and is covered by the Japanese national health insurance program.
Kinetic turbidimetric LAL analysis: An Endosafe KTA2 lysate was used to analyze samples on a sterile 96-well microtiter plate (manufacturer certified as <0.001 EU/ml). Measurements were performed using equipment provided by the manufacturer, including depyrogenated glass tubes, pipettes, and Eppendorf Endosafe pipette tips (certified <0.005 EU/ml). The analysis of each microplate consisted of duplicate wells containing endotoxin-free water as a negative control. The plate was analyzed using a Biotek ELx808 absorbance microplate reader equipped with Endoscan-V software and observed at 340 nm. The initial optical density value was set as recommended by the manufacturer to 0.03. A six-point standard curve was constructed using the initial dilution time (from 10 to 0.0025 EU/ml) of a standard dilution of a control standard endotoxin. According to the requirements of effective inference of reaction time, the correlation coefficients of all standard curves constructed were> 0.98. Due to a ten-fold dilution for plasma, the lower limit of testing is 0.025 EU/ml.
Dynamic student color LAL analysis: An Endochrome-K lysate, a pyrogen glass tube, pipette and pipette tip provided by the manufacturer were used to analyze the sample. Microplates were analyzed using a FLUOstar Omega microplate reader with MARS data analysis software, and observed at 405 nm according to the manufacturer's recommendations with an optical density value of 0.1. A standard curve was constructed using the same concentration of control standard endotoxin as described by the kinetic turbidimetric LAL assay.
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 blood|
|Testing purpose||Endotoxemia is usually reported in patients undergoing hemodialysis and is related to the pathogenesis of systemic inflammation. Blood endotoxin testing is very important.|
|Testing cycle||3-5 days.|
|Service including||We provide you with raw data and test reports.|