Ever had an Injection? Thank a Horseshoe Crab
Last week on Eureka, Dr. Christine Farrance described how environmental monitoring allows compounding pharmacies to demonstrate control over their manufacturing environments and track down the sources of any microbial contaminations that do occur. Along with sterility testing of finished products, environmental monitoring is crucial to protecting patients from infections by microorganisms.
But a sterile environment and a sterile product are not the only requirements a medicine that will be administered into the bloodstream or spinal fluid must meet. These preparations (and the components that go into them) must also be tested for bacterial endotoxins. What may be surprising to some is that this very important test is dependent on a strange, ancient creature: the horseshoe crab (Limulus polyphemus).
Bacterial endoxoins are a part of the outer cell membrane of Gram-negative bacteria. They can be present even when viable bacteria are not, is relatively heat stable, and is not removed from solutions passed through sterilizing membrane filters. Most critically, even small amounts of bacterial endotoxins can make people very sick, leading to chills, fever, low blood pressure and breathing difficulties, as well as signs of meningitis (coma, fever, stiff neck) if the spinal fluid is exposed.
The horseshoe crab’s body takes a different tack when confronted with bacterial endotoxins. Instead of fever, the horseshoe crab’s eerie blue, copper-rich blood clots when it encounters endotoxins. This allows the animal to rapidly isolate infectious agents, protecting the rest of its system. Given that this species is thought to be about 440 million years old – older than the dinosaurs – this mechanism has clearly been successful.
In the late 1960s and early 1970s scientists figured out that the horseshoe crab’s bacterial endotoxin clotting-response could be put to work in the name of drug safety. The Limulus Amebocyte Lysate (LAL) assay quickly replaced older methods of testing for bacterial endotoxins that relied on rabbits, and was approved by the FDA in 1983. In the years since, researchers have found ways to make this important test faster, cheaper and more reliable.
The key to the LAL are the horseshoe crab’s amebocytes, which are akin to human platelets. To get these cells and the valuable enzymes they contain, horseshoe crabs are collected from the Atlantic Ocean and brought to a collection facility where they are cleaned and inspected. This is followed by the bleeding process, in which about 25% of each animal’s blood is carefully removed. The crabs are then taken back to their native waters (within 24 hours) and set free. In South Carolina, the Department of Natural Resources strictly oversees the collection and release of horseshoe crabs. Every effort is taken to minimize injury to the crabs. Last year, the percentage lost to stresses of handling was estimated at approximately three percent.
Some have expressed concerns that the LAL assay will mean the eventual end of horseshoe crabs. This could not be any further from the truth. Recognition of the importance of these creatures for biomedical uses has spurred regulations that are protecting the animals from overfishing. In 2000 the Atlantic States Marine Fisheries Commission introduced trawling quotas to limit the number of horseshoe crabs caught for uses other than LAL, reducing harvest numbers by 80 percent. In South Carolina, horseshoe crabs are allowed to be caught only for the production of LAL.
Advancements in how the LAL assays is done have also taken pressure off of the horseshoe crab population. Today the tests can be conducted using specially designed cartridges that use 20 times less raw material than traditional LAL assays. These cartridges also allow bacterial endotoxin testing to be done more efficiently and robustly by removing the need for lab workers to carry out multiple sample preparation steps that can introduce errors.
These conservation efforts have helped to stabilize and in some cases increase horseshoe crab populations, which is good for the animals themselves, good for other animals in the ecosystem that depend on them for food, good for the LAL industry and, ultimately, good for patients who receive medicines that have been assured to be free of bacterial endotoxins.