Blue Blood

Limulus polyphemus, commonly known as the Atlantic horseshoe crab, is a marine arthropod. They are more closely related to spiders than to crabs. The name Polyphemus refers to the one-eyed giant in Greek mythology. It is actually a misnomer since these horseshoe crabs have ten eyes, including two lateral compound eyes that earned Dr. Haldan Keffer Hartline the 1967 Nobel prize in physiology or medicine. Today, Limulus are known for their blue blood. Unlike us, they don’t possess iron-containing hemoglobin but instead have Copper rich hemocyanin, which imparts a milky blue color to their oxygenated blood. The story of the blue blood that saves millions each year started in the 1880s with an American medical Researcher, Fred Bang. Bang was studying blood circulation using Limulus as a model when he discovered that one of his crabs died as a result of bacterial infection caused by Vibrio bacteria. The infection caused the crab blood to turn into a singular jelly-like clotted mass. His investigation revealed that even heat-treated bacteria could produce this phenomenon, and thus, the clotting was not a specific disease. He also found that the clotting resulted only in the presence of Gram-negative bacteria. He noted that this was similar to the shwartzmann phenomenon in mammals that causes thrombosis in tissues that come in contact with endotoxins

Around this time, Jack Levin, a hematologist, joined Dr. Bang’s lab. Together, they figured out that this reaction is a highly sensitive phenomenon and can act as a sensor to detect pyrogenic endotoxins. They soon discovered that it was the crab’s immune response. Being an arthropod, Limulus has a semi-closed circulatory system with open sinuses and is also cold-blooded. So, unlike us, they are more vulnerable to infections, and they can't entrap bacteria in specific areas or increase their body temperature to fight off infections. Hence, they have evolved a rapid yet rash system of detecting bacterial LPS, which is essential since the oceans are awash with microbial life, both pathogenic and beneficial. Unlike our well-endowed adaptive immune system, these crabs have to get by with only the innate immune system with a single type of immune cell called amoebocyte. These cells are obolid and are involved in wound-healing and engulfment of foreign materials. These cells contain specialized granules filled with clotting factors called coalugens that are released on exposure to endotoxins. This results in coagulation of the immediate environment, which can entrap bacteria and prevent further infection. Large incisions/wounds result in larger clots that can act as a makeshift barrier. Dr. Bang observed that these clots are stable enough to even prevent Brownian motion in the enmeshed bacteria.

This simple phenomenon doesn’t seem that paramount until you realize its potential application in the medical and pharmaceutical industry. Any drug/equipment that goes into the human body must be sterile. This includes not only pathogens but also their products like endotoxins and pyrogens. In the past, labs and Drug companies used large colonies of rabbits to detect endotoxins (Rabbit pyrogen test). Rabbits, like humans, are sensitive to endotoxins and produce fever in their presence. Thus, drugs were injected into rabbits to detect endotoxin contaminants. This method was not only expensive but also time-consuming and painful to the animals. Thus, the Limulus amoebocyte lysate (LAL) assay became a popular, albeit expensive, alternative (one liter of this costs around 10 lakh rupees). LAL assay is now the worldwide standard for testing bacterial contamination. The lysate is produced by extracting 30 percentage of total blood volume from the crabs in a non-lethal manner through their large dorsal blood sinus, and the amebocytes are separated for use. Conservationists estimate that even though this non-consensual blood donation is harmless to the crab, it leaves them weak and vulnerable, which also exerts a heavy toll on their ecosystem. LAL is currently the most sensitive and fastest pyrogen test and can produce results within 5 minutes.

In 1995, researchers from the National University of Singapore identified and isolated the gene for the endotoxin-sensitive protein (factor c). The recombinant protein version of this was produced using yeast to create a rapid endotoxin test devoid of Limulus blood. However these synthetic tests are still not widely available, and Limulus blood remains the most popular. Thus, from an antibiotic to the Covid vaccine, you have the blue-bloods to thank!

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                  Figure 3. Fred Bang

            Figure 4. An amebocyte releasing clotting factors

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              Figure 6. Extraction of the Limulus blood

      Figure 7. Jack Levin, MD, carrying out his early studies with horseshoe crabs