Discovery of LAL

The clotting properties of the horseshoe crab’s blood were first documented by W.H. Howell of Johns Hopkins University in 1885. This discovery is important to later research performed at the Wood’s Hole Marine Biological Laboratory (MBL) located at Wood’s Hole, Massachusetts. During the 1950’s, the MBL scientist Fredrick Bang discovered the component of the blood that caused clotting to occur.

The white blood cells (amebocytes) within the horseshoe crabs blood clotted when exposed to endotoxins. Later, Dr. Bang and Dr. Jack Levin developed a method for detecting endotoxins using an extract of the white blood cells called Limulus Amebocyte Lysate (LAL).

• Limulus – Taxonomic name of the horseshoe crab

• Amebocyte – The white blood cells of the horseshoe crab

• Lysate – Describes the original process used by Bang and Levin to rupture the cell membrane

In the 1970’s, the LAL test was recognized by the Food and Drug Administration (FDA) as an alternative to current methods of testing for endotoxins. Due to the efforts of Donald Hockstein, Edward Seligmann and James Cooper at FDA, the LAL test became an FDA required test for the presence of endotoxins in injectable drug products and implanted medical devices.

What are Endotoxins?

Endotoxins are fever inducing bacterial toxins found only in gram-negative bacteria. They are able to resist destruction by steam sterilization and removal by filtration. Endotoxins are found within the cell wall of gram-negative bacteria. As these cells grow and die, the endotoxins are released into the surrounding aquatic environment. Lipopolysaccharide is the biologically active component of endotoxins.

During the late 1800’s, vaccinations were being developed to combat certain diseases. In many cases, people that where given these vaccines became sick with an illness called ‘injection fever’. Years later, injection fever was discovered to be an immune response to the presence of endotoxins in two types of bacteria. Gram-negative bacteria is the one type commonly found in aquatic environments. It is called gram-negative because the bacteria do not become stained during the Gram staining process.

How the LAL Test Works

The LAL Gel Clot test is simple to use. Add a small amount of the sample solution being tested to a glass test tube containing the LAL powder. Next, incubate the test tube at 37º Celsius for 45 minutes. After time has lapsed, invert the test tube and observe the reaction.

Observation: sample remains liquid -----> Result: free of endotoxins
Observation: sample forms clot ------> Result: endotoxin present

The LAL test has several advantages over the previously used Rabbit pyrogen test. LAL delivers faster results than the 3 hours it takes the rabbit test to show results and it can detect lower levels of endotoxin.

The following medical products are typically tested for endotoxins using the LAL test: IV-solutions, dialysis tubing, pacemakers, other surgical implants, vaccines and other injectable medicines.

Another test using LAL called the chromogenic substrate method creates a color rather than a clot to detect endotoxins. The LAL reagent turns a yellow color. The degree of coloration measured after a fixed period of time indicates the presence and quantity of endotoxins in the sample.

Producing the LAL Test

Biomedical companies obtain the LAL by bleeding horseshoe crabs in a laboratory environment. Trawlers are contracted to catch adult horseshoe crabs for bleeding. Crabs are collected daily during specific seasons and brought to a nearby bleeding facility by truck. The crabs are washed to remove sand and other marine debris from their exoskeletons. Those crabs without visible injuries are placed on a bleeding rack and bled by puncturing the heart with a large gauge needle. On average, 30% of the crab’s blood is removed before the wound clots naturally.

The blood is placed in a centrifuge to separate the amebocytes from the blue haemolymph that comprises the supernatant. Distilled water is then added to the separated amebocytes. The added water will eventually cause the cells to burst, or lyse. Clotting proteins inside the cells are released and separated from the rest of the solution. The collected proteins are further processed into the powdered LAL product.

The Horseshoe Crabs are generally returned to the water within 72 hours of bleeding. Some recent studies suggest that up to 15% of bled crabs die within one year. However, there is little scientific information on the survival of bled horseshoe crabs in their natural environment.

Currently, three biomedical companies produce the LAL in the United States. They are:

Associates of Cape Cod (Falmouth, Massachusetts)

Charles River Endosafe (Charleston, South Carolina)

Lonza (Walkersville, Maryland)

The FDA regulates each company’s horseshoe crab bleeding process; however, a separate state collection permit requires the crabs to be returned to the same location they were taken from or, as of 2001, sold to the bait industry.

Photographs courtesy of Lonza

Medical Uses for Horseshoe Crabs

• Blood
• Discovery of LAL
• Other Medical Benefits