Hi Bianca. Any chance that you are asking in a wrong topic? This topic is usually concerned with plasma the fourth state of matter....as in ioninized gas...not human plasma in the cells

Yes, sorry, I realize that now. I took the topic off the question. Thanks.

Hello Bianca,

There are several points to consider on human plasma endotoxin measurement.

(1) Pretreatment is necessary for the endotoxin measurement in serum and plasma

Some people try to measure endotoxin in serum and plasma by simple dilution. However, it is seldom successful, and we do not recommend this because there are usually amidase/protease activity and serine protease inhibitors in serum and plasma. They usually cause false positives and false negatives.

(2) Contamination of the containers and other labware

Endotoxin and beta-glucan contamination is often observed in general labware, such as plastic sterile pipettes, pipet tips, test tubes, serum tubes, and blood collection tubes. If the endotoxin contamination of an item is not checked, the item should not be considered endotoxin/beta-glucan free. Some people use non-sterile containers to store serum/plasma samples, but those non-sterile products are usually contaminated with endotoxin.

(3) Type of anti-coagulant used for the preparation of plasma

For plasma preparations, an anticoagulant, such as citrate, EDTA, and heparin, is used. Citrate and EDTA are more inhibitory than heparin. More dilution or addition of Mg buffer may be needed to overcome inhibition. If you use the dilution and heating method for the pretreatment, heating decrease endotoxin potency in diluted plasma with a chelating agent. In this case, you need to add magnesium or calcium before heating. The risk of heparin is contamination of endotoxin/beta-glucan because it is a natural product.

(4) Target endotoxin concentration

Since the levels of blood endotoxin are usually low, minimum dilution is sometimes necessary to detect lower endotoxin concentration in samples. Some published papers reported very high endotoxin values in normal subjects, considering the pyrogenic dose of endotoxin, 5 EU/kg. However, they are usually not reliable because of lock of consideration of sample noise in the measurement. You need to evaluate the method very carefully, when you read such kind of papers. The endotoxin levels in normal human may not detectable.

(5) Beta-glucan interference

Beta-glucan is often found in clinical samples, and there is a possibility of beta-glucan contamination on ware used. Therefore, beta-glucan blocker buffer should be used for the reconstitution of LAL reagents.

(6) Noise in the measurement

Non-specific turbidity change can be observed in the beginnings of the measurement. You can find this noise when you observe the reaction courses of the measurement. This type of noise is often observed with high immunoglobulin, high lipid, and high hemoglobin samples.

(7) LAL test method

There are several LAL test methods, the gel-clot method, the kinetic turbidimetric method, the kinetic chromogenic method, and endpoint chromogenic method. The gel-clot is not sensitive enough and not quantitative for this purpose. The endpoint chromogenic methods should not be chosen because slight turbidity of the sample affects the measured endotoxin values. Pretreated human plasma often shows turbidity and the turbidity lifts the absorbance of the reaction mixture. This causes false positives. Kinetic methods are preferable for this purpose because of their high sensitivity. Non-specific noise may affect the measurement with the kinetic methods too.

I hope this information is helpful for you.

Unformtunately, there is no satisfactory method for blood endotoxin measurement right now.

Best regards,

KAZ Tsuchiya

Thank you very much, Masakazu and Christopher for the very helpful comments.

Bianca, 

I tried all the steps Kaz listed above over a course of three months. Save your time and money and don't try to measure endotoxin in plasma or serum samples using the LAL kits. Lonza also has a different approach using a flurometric "recombinant factor C" which is also useless with biological samples. Attempting spike-recovery with any troubleshooting step never improved the erroneous result. 

We'll simply have to wait until a manufacturer optimizes a kit with the specific objective of measuring in blood samples, until then, Kaz offers sound advice; " Unfortunately, there is no satisfactory method for blood endotoxin measurement right now."

Good luck,

Patrick

Hi

Which one you recommend better for endotoxin measurement, plasma or serum?