Detecting Endotoxin: Is LAL assay based on absorbance a sustainable approach?
Based on appearances you probably would not expect this creature to hold the key to ensuring the safety of a multitude of medical devices and drugs that are currently on the market. The horseshoe crab not only looks bizarre it is quite literally from another time – 200 million years ago! It definitely earns the description as a ‘living fossil’.
To understand the importance of the horseshoe crab in medicine you must look beneath the surface, specifically at its blood –beyond the fact that it is light blue in color. The medical interest in horseshoe crab blood is based on the presence of cells called amebocytes. These cells are vital in the survival of the horseshoe crab as they serve as the key to their innate immunity against fungi and gram-negative bacteria. When the amebocytes come in contact with the endotoxin which is found in the cell walls of these intruders a cascade of events results in the coagulation, immobilization and neutralization of the pathogen.
The ability of amebocytes to produce a visible response to the presence of endotoxin is the basis for the Limulus amebocyte lysate or LAL test. We have previously reported on one form of endotoxin test in the Application Note 237: Lonza’s kinetic kit for endotoxin detection using BMG LABTECH’s microplate reader and MARS data analysis but the LAL test remains the gold standard. The LAL is the only accepted method to ensure safety of: recombinant or intravenous drugs, biologicals (such as insulin) and medical implants (such as heart valves). In addition, the LAL test is used in environmental testing of air and water samples for endotoxin.
The approach is quite straight-forward and truly only requires an absorbance based reader to detect changes in turbidity measured at a wavelength of 340 nm. All of our readers that are equipped for absorbance are up to the task of reading the LAL assay. Here are some spectral data collected over time by our UV/vis spectrometer on the CLARIOstar:
MARS data analysis software can also be used to perform kinetic calculations. For this test comparing the time it takes each sample to reach a certain OD threshold is important and is used to compare samples of known concentrations and create a standard curve that is used to calculate unknowns.
Unfortunately, production of the LAL kits is dependent on a rather crude process which involves the capture of horseshoe crabs (over 500,000 in 2015 alone) so that some blood can be collected. The blood draw volume can be from 50 to 400 mL dependent on sex and size. The blood is centrifuged to concentrate the amebocytes which are then lysed to release the coagulation proteins. Although the horseshoe crabs are able to be released after bleeding a certain number (10 – 30 %) are known to perish during the transport and/or blood extraction process. Further complications may occur leading to death after release which would not be directly associated with LAL kit production.
Another consideration is that horseshoe crab capture is occurring during mating as this is the time when they come ashore. It is unclear what effect the collection has on the reproductive capacity, especially among captured females. What is clear is that the numbers of horseshoe crabs is in decline and human collection for LAL testing is certainly a contributing factor.
Efforts are underway to ensure better practices in the collection of the crabs including identifying the best possible mechanisms of transport, including understanding the best temperatures to use and limiting the time they are out of water. Limiting the capture of the females of the species has also been proposed as a way to promote higher reproductive capacity. Furthermore, a better understanding of the impact of the bleeding process must be achieved. This needs to include how much blood can be safely drawn from an individual of a given size. Unfortunately, ‘ranching’ of horseshoe crabs has thus far proved extremely unsuccessful so capture of wild crabs will continue so long as there is demand.
The need for endotoxin testing shows no signs of decreasing and in fact there are fears that demand may increase. Species of horseshoe crab are also found in Asia and used in a similar TAL assay in those countries. Decreasing populations of the Asian horseshoe crab could lead to increased demand for the LAL assay.
Thus far there has been resistance to adopting any of the alternative methods to detect endotoxin. This is understandable for the fact that approval of any process used in safety testing is (understatement alert) difficult. But for those seeking alternatives they are definitely available. For instance, recombinant factor C (rFC) has been cloned from the Singapore horseshoe crab and is promising because it is more sensitive than the LAL assay and has a better specificity for endotoxin. The PyroGeneTM assay produces a fluorescent signal based on rFC activation. This assay should be fully compatible with any of BMG LABTECH reader that can detect fluorescence (Omega series, CLARIOstar and PHERAstar FSX).
If you would like to find out more about the horseshoe crab please check out this recent review:
The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sustainability by Jordan Krisfalusi-Gannon and his colleagues in North Carolina and New York.
It was the primary resource for this post.