The determination of bacterial endotoxins: How to avoid the contamination of the samples during handling

The handling of the samples to which the Limulus amebocyte lysate (LAL) test will be applied plays a key role in the results obtained. Bacterial endotoxins are the most frequently encountered pyrogen in the samples of medicine, foods and other products. Therefore, it is very easy for the samples that are being studied to be contaminated in the laboratory due to wrong handling. This results in erroneous results regarding the purity of the sample in question. When one would like to check that a product is free from pyrogens, the LAL test is conducted. This is because, although the pyrogen test on rabbits is sensitive to other pyrogens which are different from endotoxins, positive results are not obtained without having a positive result in the LAL test and, on the contrary, it has been seen that samples contaminated by endotoxins in small quantities can provide negative results in the test on rabbits. Through the LAL method, one can quantify the amount of endotoxins that are present. Due to these characteristics, the LAL test has prevailed not only in the industry, but also in the laboratories where research, such as the best method for the determination of pyrogens, is being conducted.

In order to conduct the LAL test, an endotoxin pattern is required, which needs to be handled with extreme care as it is the infectious agent at a high concentration in the dissolution of reference. Therefore, the first risk to consider is the one posed against the health of the person who conducts the test as well as other people that could be sharing the workspace with that person. The standard endotoxin of reference should be handled according to the instructions that come in the packaging to avoid errors when making the calibration curve.

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Before the operator starts the analysis of samples via LAL, each one of the dissolutions which constitute the calibration curve must be measured four times and the statistical analysis of these dissolutions must be prepared. One of the points to take into account is that the samples which will be subject to the LAL test should be pasteurized or freeze-dried for their preservation and never frozen as freezing leads to the loss of endotoxins which are responsible for the production of the coagulation of the hemolymph of the Limulus Polyphemus crab and for obtaining a measurable signal proportional to the quantity of endotoxins present.

All the instruments which will be used for the test of endotoxin determination must first be depyrogenated, for which any of the methods that are valid for this purpose can be used. The most common method for depyrogenating laboratory material is treatment with dry and hot air. As endotoxins are resistant to heat, temperatures higher than 250 ºC should be used and it should be checked if the material is really rendered free from pyrogens. It has been reported that, with lower temperatures and more time of exposure to heat, the instruments are also rendered pyrogen-free, for instance at 180ºC, being heated for 3 hours. There are authors that consider that autoclaving can be used for depyrogenation or acid-based hydrolysis, given that the lipid complex A - the protein that constitutes endotoxin - is ruptured by washing the material with an acid dissolution or by using alkaline. However, it has been confirmed that these methods present less effectiveness than the use of dry and hot air.

There are other methods for depyrogenating dissolutions or samples that are going to be used in research with medicine or other products that require the researcher to ensure that they are free from endotoxins. Some of these methods are: ultrafiltration, where the passage of big molecules, such as lipopolysaccharides, is prevented by making the sample pass through an ultra-thin filter; ion-exchange chromatography, where we take advantage of the fact that the endotoxins present a negative load so that they are held in the column, but, in this case, their effectiveness depends on the pH, the speed of the elution of the column and on other factors, which make the method unreliable; reverse osmosis or distillation.

We need to take into consideration that the material which is bought in a sterile state on many occasions has to be prepared for the LAL test before being used. Therefore, the use of vials and other specific glassware is recommended for the determination of endotoxins, such as pipette tips and tubes for the gel clot reaction that Wako offers in its catalogues, belonging to the BioClean® series. It is not advisable to reuse the material which has been used for a LAL test for future determination procedures or the rest of the laboratory material that could contain chemical or biological residues after being cleaned, which would hide the endotoxins and lead to false results in the LAL test.

In the LAL test, other precautions must be taken, such as letting the vials dry at least for one night if the endotoxin is applied directly at the base of the vials to measure the calibration curve and conducting this procedure in a cabin with unidirectional air current. To conduct the LAL test for all raw materials as well as water and glassware is important in the cases where sample contamination by endotoxins is observed in the final product. Moreover, each time that the LAL reagent is bought, its sensitivity with the standard endotoxin pattern should be checked and we should thus make sure that it conducts the measures correctly.

All in all, researchers and technical personnel in charge of conducting this test are recommended to work with high-quality reagents whose use does not require additional laboratory material. The kits marketed by the company "Wako" comply with these characteristics. These kits work with the use of the three methods of detection employed in the LAL test, which are the colorimetric method, the turbidimetric method and the gelation method.

Bibliography:

1) Novitsky, T. J., Oceanus, 27(1), 13-18, 1991.

2) Tsuji, K. et al., App. and Env. Microbial, 36, 705-719, 1978.

3) Sandle, T., American Pharm. Reviews, epub 28 octubre, 2013.

LAL PRODUCT LINE

Control Standard Endotoxin	Endotoxin Extracting Solution for LAL Test	PYROSTAR™ ES-F / Plate With CSE