Welcome to the third, and final, part of the saga of horseshoe crab blood.
But, it is now time to ask and answer some difficult questions about the past, present and the future of this test.
How do we get the blood needed to make LAL?
There are many companies that extract horseshoe crab blood and there may be slight differences in how each of them does it. But, in general, the horseshoe crabs are captured during their annual mating season, by hand or trawlers, transported in crowded conditions and kept outside their natural environment for 24-72 hours. By puncturing the heart with a needle, upto 30% (about one-third) of each horseshoe crab’s total blood volume is extracted. They are then returned back to the ocean.
How does this affect the horseshoe crabs?
Many scientific studies have shown that horseshoe crabs are negatively affected by the biomedical bleeding process. Besides a mortality rate of up to 30%, bled horseshoe crabs moved more slowly, and showed overall lower activity. Compounding all this is the fact that they are harvested primarily during their spawning/mating season, and females are preferentially harvested due to their relatively large size. This means that all the negative effects happen more often to females (that are already invested heavily in nesting and producing eggs) and also happen at the only time of the year when the horseshoe crabs have a chance to reproduce.
What did we do before the LAL test?
Any biological molecule that causes fever is called a pyrogen. Scientists use this term to describe the causative agents of ‘injection fever’. Back in the old days, doctors and scientists found that injecting many substances into the human body resulted in the development of fever and/or hemodynamic shock (loss of blood pressure and multi-organ failure, leading to death). Note: Subsequent research showed that endotoxins are an important type of pyrogen.
Starting in the 1940s, scientists settled on using rabbits to test for the presence of pyrogens. Any drug solution that needed to be tested was injected into rabbits. Then, they were monitored to see if they developed a fever (or worse symptoms).
But there are problems with the rabbit test…
Even though the rabbit pyrogen test is a useful test, it has a number of problems of which I will list four important ones. Firstly, it is a qualitative (pass or fail) rather than a quantitative test (how much toxin is present). Secondly, it is not sensitive enough. It cannot detect pyrogens at the lowest concentration which can cause fever in humans. Thirdly, since it involves the use and monitoring of live animals, there is considerable variation because of the age, gender, housing and handling conditions of the animals. Finally, it is a time-consuming test that takes about three hours to complete.
Why is the LAL test a better alternative?
The LAL test is more sensitive. It can detect endotoxins at much lower concentrations than the rabbit test. Being a quantitative test, it can be used to measure the level of endotoxin contamination in a sample. Since the test procedure itself does not involve the use of animals (it is done using molecules extracted from horseshoe crab blood), it requires lesser money and human effort. Also, it is relatively a very fast test (the latest version only requires 15 minutes).
But the LAL test also has problems …
Certain drugs and biological samples interfere with the LAL test. Also, it can detect only endotoxins and cannot detect other kinds of pyrogens.
Besides these direct problems, there is also the issue of the source of LAL. Given the mortality and other adverse effects that bled horseshoe crabs show, a substantial increase in the demand for the LAL assay might lead to irreversible endangering or extinction of the horseshoe crab. This could also trigger problems in other organisms that are ecologically linked to horseshoe crabs.
So, there are clearly problems with both the rabbit test and the LAL test. The rabbit test is not very dependable and the collection of horseshoe crab blood cannot remain sustainable for long. So, what is the solution?
Can genetic engineering and tissue culture make things better?
There is some consensus among the countries that have access to horseshoe crabs in their territories. In most of the countries where there is enough data, horseshoe crab populations have been shown to be depleted to varying degrees. The factors that affect horseshoe crab populations are harvest pressure, habitat loss and environmental pollution. Even if we can reduce habitat loss and environmental pollution, horseshoe crabs will continue to be harvested for LAL unless acceptable alternatives are adopted.
Recently, scientists used genetic engineering to manufacture the enzymatic molecule that is central to the LAL test. The gene coding for the molecule is inserted into lab grown cells of different types (yeast, insect, mammalian), which then express the necessary molecule. This molecule is subsequently extracted and purified. Known as rFC (recombinant Factor C), this molecule is crucial to the development of the latest version of endotoxin testing.
Besides genetically engineered rFC, scientists are researching ways to synthetically produce the blood and amoebocytes of horseshoe crabs, through artificial tissue culture methods. There are also some pyrogen tests under development that can be done with human blood and/or lab-grown cell lines derived from human tissue.
Image: Cartoon showing endotoxin test types and sources; adapted from multiple references cited at the end of the article
These new forms and sources of endotoxin tests require further standardisation and/or wider acceptance. Nevertheless, there is at least one viable alternative (rFC) that can minimise and perhaps end the bleeding of horseshoe crabs for endotoxin tests.
Up to this point in the LIR series, we have looked at barnacle adhesion, the importance of blood clotting in the horseshoe crab’s immune system, and its use in endotoxin testing. In my next LIR article, we will look at the complex relationship between immunity and reproduction in living organisms.
Novitsky, T. J. “Discovery to commercialization-the blood of the Horseshoe-crab.” Oceanus 27.1 (1984): 13-18.
Anderson, Rebecca L., Winsor H. Watson III, and Christopher C. Chabot. “Sublethal behavioral and physiological effects of the biomedical bleeding process on the American horseshoe crab, Limulus polyphemus.” The Biological Bulletin 225.3 (2013): 137-151.
Leschen, A. S., and S. J. Correia. “Mortality in female horseshoe crabs (Limulus polyphemus) from biomedical bleeding and handling: implications for fisheries management.” Marine and Freshwater Behaviour and Physiology 43.2 (2010): 135-147.
Hort, Edward C., and W. J. Penfold. “The relation of salvarsan fever to other forms of injection fever.” Proceedings of the Royal Society of Medicine 5.Pathol Sect (1912): 131.
Hartung, Thomas, et al. “Novel pyrogen tests based on the human fever reaction.” ATLA-NOTTINGHAM- 29.2 (2001): 99-124.
Kreamer, G. 2012 Biomedical use of horseshoe crabs slides for classroom use (including teacher notes and references). Delaware Division of Fish and Wildlife, Smyrna, DE.
Tanacredi, John T., Mark L. Botton, and David R. Smith, eds. Biology and conservation of horseshoe crabs. New York, NY: Springer, 2009. 465-475.
Ding, Jeak Ling, and Bow Ho. “Endotoxin detection–from limulus amebocyte lysate to recombinant factor C.” Endotoxins: Structure, Function and Recognition. Springer Netherlands, 2010. 187-208.
Joshi, Bhupali, Anil Chatterji, and Ramesh Bhonde. “Long-term in vitro generation of amoebocytes from the indian horseshoe crab Tachypleus gigas (MÜLLER).” In Vitro Cellular & Developmental Biology-Animal 38.5 (2002): 255-257.