Life is Rough (02) – The saga of horseshoe crab blood, part 1

In the previous article of the ‘Life is Rough’ series, we looked at how blood clotting allows barnacles to stick as well as they do. Through this article, and more of them in the coming week, we will continue to talk about blood clotting but, for a different reason and, in a different organism: the horseshoe crab.

LAL

GIF by Shruti Muralidhar

‘Horseshoe crab’ is the commonly used name for an animal that looks somewhat like a crab with a tail, that is wearing a large helmet. But it is more closely related to spiders and scorpions than it is to crabs. It lives in the sea, eats small marine creatures like worms and clams and, comes to sandy shorelines to reproduce.

horseshoe_crab

Image source: https://www.flickr.com/photos/jsjgeology/24522890902

Limulus polyphemus  – Atlantic horseshoe crab in Florida, USA.

There are four known species of horseshoe crabs, one of which lives along the east coast of Central and North America, while the other three live along the coastal regions of some Asian countries. By 1964, scientists had produced an extract from the Atlantic horseshoe crab’s blood cells that was responsible for blood coagulation in the presence of bacterial endotoxins. Subsequently, they found that the extract was sensitive enough to be used as a test to detect these same toxins. The test is currently so sensitive that it can detect femtogram levels of endotoxin. For reference, 1 femtogram is equal to 0.000000000000001 grams.

This test, called the Limulus Amoebocyte Lysate (LAL) test, has been used since the early 1980s as a safety requirement for a variety of pharmaceutical drugs and implants. It continues to be the most trusted and sensitive test to detect these toxins, having been approved by the FDA, as well as similar organisations in other countries. Note: When manufactured from other species of horseshoe crabs, the test is designated TAL or CAL but it works just like LAL.

What are these bacterial endotoxins and why do we need to check for them? Endotoxins are molecules in the outer membrane coat of certain bacteria. These toxins are released in small amounts while the bacteria are alive, and in large amounts when they die. If these toxins are released into the human body, anywhere other than in the gastro-intestinal tract, bad things start to happen. At sufficient quantities, these toxins can cause a range of symptoms including fever, weakness, loss of blood pressure and even multiple organ failure leading to death.

Common methods of sterilising materials and solutions use heat treatment and filtering. But, endotoxins pass straight through filters that block bacteria and are resistant to the level of heat sufficient to kill bacteria. Ironically, killing bacteria only produces more endotoxins. So, just sterilising something does not mean that it is endotoxin-free. It has to be heated to much higher temperatures, to destroy the toxins. This is why we need to check for them and this is why the LAL test is so important. Pharmaceutical companies depend on it to ensure that their injectable products and implantable devices do not contain dangerous levels of endotoxin.

So, how is the blood of the horseshoe crab turned into LAL? Here’s a fun cartoon by Shruti Muralidhar that outlines the basic process.

LAL_1.png

The saga of the horseshoe crab and how we use its blood has just begun. Stay tuned for my next articles, where I will talk about how the horseshoe crab blood is sourced, the immune system of the horseshoe crab, the nuts and bolts of the clotting mechanism, how horseshoe crab blood replaced rabbits for endotoxin testing and even how the LAL assay went to the International Space Station. Stay tuned!

References:

https://www.ncbi.nlm.nih.gov/pubmed/20593268

https://www.ncbi.nlm.nih.gov/pubmed/17727802

http://ajpgi.physiology.org/content/290/2/G377

http://www.tandfonline.com/doi/abs/10.1080/20026491051677

Novitsky, T. J. 1984. Discovery to commercialization: the blood of the horseshoe crab

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.

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