For the Limulus test, attention should be paid not only to the Limulus reagent (LAL), but also to other elements such as the water used for the bacterial endotoxin test. In this episode we would like to elaborate on the use of plastic consumables as containers for endotoxin and samples, pipette tips for dispensing reagents, etc.

Labware for LAL tests should meet the following conditions:

1. Labware should not be contaminated by endotoxins or other LAL-reactive substances
2. Endotoxins and other samples should not adhere to the labware
3. Labware should not affect endotoxin activity

LAL tests have conventionally been performed with glass labware that has been subject to dry heat sterilization at 250°C or higher for an appropriate period of time¹⁾. The foremost reason for this preference in material is that the heat treatment for sufficiently inactivating endotoxin is easy for glassware.

Containers made of a certain type of soda lime glass could impact endotoxin activity. However, this effect is not observed with borosilicate glass such as Pyrex. Although it has been suggested that endotoxins may adsorb onto glassware²⁾, this doesn't seem to be a problem when carefully cleaned glassware is used.

Consequently, glass has been the standard material of choice for LAL test equipment; however, disposable, sterilized plastic labware has come to be used for reasons such as convenience.

Plastic labware cannot be subjected to dry heat sterilization to inactivate endotoxins, so products must be free of endotoxin contamination at the time of manufacture. Fortunately, endotoxins are undetectable in most sterilized disposable plastics. That said, few products are guaranteed to be endotoxin-free and those making this claim need to be approached carefully because contamination may be detected with highly sensitive measurements.

In other words, endotoxin contamination of labware should be confirmed with the measurement technique to be carried out. Although water or saline is commonly used to extract contaminants from labware, this is believed to be risky. We discovered that endotoxins adsorbed to plastic can be detected with solutions containing proteins such as albumin, but not with water or saline³⁾.

This type of contamination is not an issue when measuring samples such as water but could be troublesome when measuring plasma and other samples containing proteins, which is a subject that will be discussed in another post.

Adsorption of endotoxins onto container surfaces becomes a concern when endotoxin solutions are formulated or preserved with plastic labware. Endotoxins in aqueous solutions are less likely to adsorb to polystyrene, but can adsorb to polypropylene. Therefore, labware made of polypropylene should be managed with care. Adsorption characteristics will vary depending on the labware manufacturer and production lot, and can change due to the coexistance of proteins and other substances.

Although there have been no reports of plastics interfering with endotoxin activity through factors other than adsorption, there remains the possibility that activity can be influenced by stabilizers and plasticizers used during production. When we observed enhanced endotoxin activity in water that was stored in a container made from a certain type of polystyrene, we speculated the cause to be eluate from the material.

Accordingly, it is essential to confirm that plastic labware will not contaminate or otherwise impact the measurement. Such confirmation should be made under the working protocols for each type of equipment. This is not to say that all glassware is safe, but it is recommended to manage plastic labware judiciously while keeping in mind that glass is generally the accepted standard.

References

1. Guide to the Japanese Pharmacopoeia 12th Edition (B-529). (1991). Hirokawa-Shoten.
2. Eighth Supplement, USP22-NF17, p.3349-3350, (Pharmacopeial Convention Inc., MD) (1993).
3. Proceedings of the 6th Annual Meeting of the Japanese Society for Alternatives to Animal Experiments (pp. 110-111). (1992).