Horseshoe Crabs Prove Lucky for Drug Tests

Researchers seeking to eliminate potentially deadly contaminants from drugs are relying increasingly on an unlikely source of help: the blue blood of horseshoe crabs.

An extract from the crab blood is the base ingredient in chemical tests that can detect and measure contaminants at levels as low as one part in a billion. These contaminants produce sudden, severe fevers that can be fatal in patients already weakened by disease.

The test is so effective that it is gradually being adopted as the official standard for ensuring the purity of various classes of drugs, said Aubrey Outschoorn, a senior scientist with the U.S. Pharmacopeia, the standard-setting body for drugs in the United States.

“Up to now, there has been, in many cases, no test” to detect the contaminants, Outschoorn said. The contaminants, bacterial byproducts called endotoxins, are found in a wide variety of drugs. They are not destroyed by sterilization and cannot be completely eliminated by chemical purification, he said.

Rabbit Test

The only other way to detect endotoxins has been to inject drugs into rabbits and then watch for signs of fever, Outschoorn said. But the rabbit test could not be used with all drugs, he said.

The horseshoe crab extract--known as limulus amoebocyte lysate--enables endotoxin testing to be done faster and perhaps more cheaply, but those are not its principal advantages, Outschoorn said.

“Its great virtue is that it is more sensitive than the rabbit test, and therefore it can afford a bigger margin of safety for patients than the rabbit test can ever do,” he said.

Associates of Cape Cod, a small company in Falmouth, is one of the major producers of the extract. In the summer, when horseshoe crabs are easily gathered on beaches or in shallow water, a small van backs up to the company’s side door early each weekday morning.

Student Employees

Inside the van are several hundred damp, wriggling horseshoe crabs packed in plastic garbage cans. The crabs, collected from Pleasant Bay, off Cape Cod, and Narragansett Bay, R.I., are hustled inside where college students, using sterile needles, extract a small vial of blood from each crab.

The crabs, apparently no worse off, are returned to the garbage cans. By noon they are on the way back to the cool Atlantic waters.

The unique ability of the horseshoe crab to detect bacterial endotoxins was an inadvertent discovery at the Marine Biological Laboratory in Woods Hole, four miles down the road.

Researcher Frederik Bang was exploring the ability of the crab to resist infections, even though it lacks the kind of protective immune system that higher animals have, said Stanley Watson, a microbiologist at the Woods Hole Oceanographic Institution and the president of Associates of Cape Cod.

“If we went swimming in Eel Pond,” he said, referring to a saltwater inlet behind the laboratory, “we’d be dead in a week without antibodies.

“But here are all these invertebrates without antibodies. We were all interested in how they survive disease.”

Bang discovered that the crabs’ blood clotted around any invading bacteria. This is presumably how the crabs protect themselves from infection, Watson said.

Triggering the Process

Bang determined in the mid-1970s that the clotting reaction was triggered by bacterial endotoxins, even in very small amounts.

That reaction is the basis of current tests. A small amount of a drug is mixed with water and the crab-blood extract, a freeze-dried white powder. Depending upon the test used, the mixture will either clot, become cloudy, or turn color if endotoxins are present.

The test is so sensitive that it can detect endotoxins in concentrations as small as one part per billion, said Ronald Berzofsky of Whittaker M.A. Bioproducts in Walkersville, Md., another leading producer of the crab-blood extract.

The U.S. Food and Drug Administration is rewriting certain guidelines to allow wider use of the extract, said spokesman Terry Munson. Regulations still require rabbit tests for most drugs to be certified as pure, but those regulations are changing.

Water for Injection

Water meant for injection in combination with drugs, for example, can now be released to the public on the basis of a crab-extract purity test alone, and so can certain radioactive drugs, Munson said.

Researchers now believe that the limulus test may eventually find new uses in diagnosing spinal meningitis and certain bacterial infections in the blood, though some components in the blood confound the test.

“There are a lot of problems in the medical applications,” Watson said. “I think it will be important in the future.” The test seems to be especially suitable for use in detecting urinary tract infections and spinal infections, such as those that cause meningitis, he said.

Environmentalists on Cape Cod have raised questions about whether increasing demand for the extract could devastate the population of horseshoe crabs--technically not crabs at all, but relatives of spiders and scorpions. Associates of Cape Cod uses about 50,000 crabs a year.

So far, it appears that taking blood from the crabs will not seriously affect their population; most of the crabs seem to survive when put back into the ocean, said Mark Botton, a marine biologist at Fordham University in New York.

“From what I can tell, the (limulus antibody) people are very concerned with maintaining populations,” he said. “If they bleed an animal and it’s released and caught a second time, they can recognize that (because of a scar left by the needle), and they will not bleed an animal that they have previously taken blood from.”