AWARDEES: Jack Levin and Frederik Bang

SCIENCE: Limulus Amebocyte Lysate (LAL) Test

FEDERAL FUNDING AGENCIES: Atomic Energy Commission, National Institutes of Health, U.S. Public Health Service

The Blood of the Horseshoe Crab: Its Improbable Contribution to International Public Health

The horseshoe crab has been described as an animal that “time has left behind.” And it’s no wonder that these marine creatures have been referred to as living fossils — they’ve been around for more than 450 million years, making their appearance 100 million years before the dinosaurs.

But there’s a reason why these primitive arthropods that are more closely related to spiders and ticks than crabs have survived for such a long time: their immune system.

Starting Off with a Bang

The Atlantic Ocean is one of the few places horseshoe crabs are found in the world today. Limulus polyphemus, the scientific name for the Atlantic horseshoe crab, can be found in areas stretching from the Gulf of Mexico up to the northeastern United States, where pathobiologist Dr. Frederik Bang spent summers performing research at the Marine Biological Laboratory (MBL) in Woods Hole, MA.

Frederik “Fred” Bang chaired the Department of Pathobiology at the Johns Hopkins University School of Public Health. He received his M.D. from the Johns Hopkins School of Medicine in 1939 and was a pioneer in applying marine biology to medical research. Dr. Bang’s many interests included using invertebrates to study biological phenomenon; he believed that studying “transparent” animals — those in which circulation could be observed in the intact animal — could lead to a greater understanding of the physiological processes in vertebrates, such as humans.

In the early 1950s, Bang was studying the circulatory system of the horseshoe crab and its response to bacterial infection at the MBL. Funded by the U.S. Public Health Service, he would inject bacteria obtained from fresh sea water into horseshoe crabs of varying sizes and study their reaction. Horseshoe crabs have evolved an incredibly efficient and potent mechanism to fight bacterial infections; when the animal is injured, its blood — which turns blue when exposed to oxygen due to the presence of a copper-rich protein called hemocyanin — seals off the infected area with a small clot, stopping the entry of further bacteria. But one day, Bang noticed that one of his crabs died from an unknown infection that had caused nearly the whole volume of blood in the crab to clot into a semi-solid mass.

No other bacteria had caused such a reaction.

When he cultured the bacterium from the first animal and injected it into other horseshoe crabs, they also experienced intravascular clotting and died. Investigating further, he found that only “Gram-negative” bacteria produced this reaction. Gram-negative bacteria cause infections such as pneumonia and meningitis, but the same reaction occurred whether the Gram-negative bacteria were heat-treated or not, so he realized that live bacteria were not required to cause the horseshoe crab’s blood to clot.

He published his findings in 1956 and put aside his initial observations for nearly 10 years.

A Tale of Two Professors

In 1963, while discussing data from the project, a colleague at Johns Hopkins convinced Dr. Bang that collaborating with a hematologist on the project could help solve the mystery. The colleague, Dr. C. Lockard Conley, who headed Hopkins’ Hematology Division, recommended a research fellow from his lab, Dr. Jack Levin.

Levin recalls that “It was just two professors at the same school talking to each other. It’s a real lesson in how one’s professional career can hinge on totally unexpected events over which you have absolutely no control.”

Jack Levin received his M.D. from the Yale University School of Medicine in 1957. In the intervening years, he served as a lieutenant in the U.S. Public Health Service and was a clinical associate at the National Cancer Institute.

As a hematology research fellow at the Johns Hopkins School of Medicine, he was studying the Shwartzman Reaction — a rare reaction of a body to particular types of toxins called endotoxins — including altered platelet function. Specifically, he was investigating the effects of bacterial endotoxins on blood coagulation (clotting) in rabbits. Endotoxin is a key component in the cell wall of all Gram-negative bacteria; it can be hard to detect and is resistant to drugs. Exposure to a high amount of bacterial endotoxin is dangerous to humans. Levin’s initial work was funded by the U.S. Atomic Energy Commission. In the midst of the Cold War, the U.S. was interested in learning more about platelets and hemorrhage.

Levin had never heard of, much less seen, a horseshoe crab before. But he agreed to spend the summer at the Marine Biological Laboratory in Woods Hole to study the similarities between Limulus amebocytes, the only type of circulating blood cell in the horseshoe crab, and human platelets.

He was able to demonstrate early on that cell-free plasma from a horseshoe crab would not clot. While trying to study the blood cell, he kept having trouble keeping the blood from clotting. Samples that were fine when he left the lab at night were coagulated when he returned in the morning, and none of the anti-coagulants on the market made any difference.

An Accidental Aha Moment

Baffled by this phenomenon, he considered the possibility that the samples might be contaminated by bacteria or some sort of bacterial component. In desperation, he applied what he had learned from his other work on endotoxins and blood coagulation in rabbits and prepared new samples in sterile, endotoxin-free glassware. Amazingly, the blood did not clot.

At that moment he recognized that he had identified a blood-clotting mechanism that was responsive to bacterial endotoxin. With this realization, Levin was able to learn more about the clotting and show that the entire blood coagulation mechanism in Limulus was contained in the amebocytes and was extremely sensitive to the presence of endotoxins. “I think only an investigator who was working with endotoxin would have ever considered the possibility that endotoxin was causing Limulus blood to clot,” Levin says.

Using this knowledge, Levin created the Limulus amebocyte lysate (LAL) test, which could test for bacterial endotoxins using horseshoe crab blood. The lysate was prepared using a non-lethal method of obtaining blood from the crab’s pericardial sinus, and the resulting test was much more sensitive than the only available test for endotoxins at the time, the Rabbit Pyrogen Test.

Rabbits, like humans, are sensitive to endotoxin. And as such, all parenteral (injectable) drugs were required by the Food and Drug Administration to pass the Rabbit Pyrogen Test before they could be approved for use. The majority of pyrogens — bacterial contaminations in parenteral drugs that can cause a fever — in the pharmaceutical industry are endotoxins, and so passing this test was meant to show that a drug did not contain concentrations of endotoxin sufficient to produce fever.

The Rabbit Pyrogen Test however, was a costly, inefficient and often inaccurate process. For the test, a sample would be injected into a group of rabbits. If that sample caused sufficient fever in those rabbits, the drug was ruled contaminated, and it failed the test. But if no fever presented within the next 4-6 hours, then it was concluded that there was no significant contamination and the drug was safe. Pharmaceutical companies had to house thousands of rabbits in order to perform these tests.

The LAL test can return a result in as little as 45 minutes and can detect the presence of endotoxins at levels of less than one part per trillion. So, when Levin realized that he had a very sensitive in vitro assay, it became apparent that he had a highly suitable substitute for the rabbit test.

Although the LAL test was first described in 1965, it took almost 20 years before the test was formally approved as an end-product endotoxin test by the FDA due in part to resistance to the new, more sensitive test from pharmaceutical companies.

Today, the LAL test is the standard screening test for endotoxin contamination worldwide, with approximately 17.5 million samples tested (amounting to roughly 70 million tests performed) using this method each year, according to Jack Levin. It is used commercially to test all intravenous fluids, parenteral drugs, and implantable medical devices before they are used in patients. Overall, revenue from the industry is estimated to be approximately $250 million annually. There have been no FDA-confirmed pyrogenic outbreaks due to a false-negative result from the LAL test in the over 40 years of its use. The LAL test has been named one of the “100 Most Important Contributions to Public Health” by the Johns Hopkins Bloomberg School of Public Health.

A Lasting Legacy

Fred Bang spent 35 years as a member of the faculty of the Johns Hopkins University, including as Chairman of the Department of Pathobiology from 1953 until his death in 1981. His extensive work at the university ranged from electron microscopy to parasitic diseases, and his field-based efforts led to the establishment of Hopkins’ Centers for Medical Research and Training in Calcutta, India and Dacca, Bangladesh, which he directed from 1961 to 1976. Jack Levin remembers Fred Bang as a Renaissance man – “nothing he saw failed to interest him.”

Jack Levin served on the faculty of Johns Hopkins University School of Medicine until 1982, when he transitioned to the University of California School of Medicine, San Francisco. While at Hopkins, he was the inaugural recipient of the Frederik B. Bang Award for Research in Bacterial Endotoxins. Levin is currently a Professor of Laboratory Medicine and Medicine at the UCSF School of Medicine and has remained a member of the Marine Biological Laboratory, where he served a term as a Member of the Board of Trustees. In 2014, the Parenteral Drug Association recognized him with a special award on the 50th anniversary of the discovery and description of the LAL test.

“Serendipity is an unexpected observation combined with a mind that is prepared to consider and interpret the observation.” – Dr. Jack Levin