If you have ever had an injection before, chances are that injectable drug or vaccine was tested using the blood of the horseshoe crab shown above.

Bacterial endotoxins, also known as lipopolysacharides (LPS), are molecules that make up an important part of the gram negative bacterial cell wall. These molecules are responsible for structural integrity of the cell wall, protection from certain kinds of chemical attack, and maintenance of the cell membrane negative charge.

LPS are also an important virulence factor, and are secreted by gram negative bacteria through the membrane vesicle trafficking system, which releases these molecules, other proteins and virulence factors into the environment.

When endotoxin is released into the human blood stream, the LPS bind the CD14 receptor complex on many immune cells, including monocytes, macrophages, and B cells. Upon binding, these cells secrete pro-inflammatory cytokines, nitric oxide, and other immune response molecules.

This causes the body to go into septic shock, characterized by a drop in blood pressure, fever, and widespread blood clotting which can often result in death.

Thus, testing for the presence of endotoxin is of prime importance for pharmaceuticals and compounded sterile preparations. Any injectible drug that contains endotoxin could pose a dangerous threat to patient health.

Three types of endotoxin tests exist: the chromogenic method, the turbidimetric method, and the gel clot method. The gel clot method is considered the gold standard and believed to have the most accuracy and sensitivity.

The gel clot method uses blood from the Horseshoe crab ( Limulus polyphenus ). Unlike vertebrates, horseshoe crabs do not have hemoglobin in their blood, but instead use hemocyanin to carry oxygen. Because of the copper present in hemocyanin, their blood is blue. This blue blood contains amebocytes similar to white blood cells of vertebrates in defending the organism against pathogens.

These blood cells are used to make Limulus Amebocyte Lysate (LAL) , which is used for the detection of bacterial endotoxins in medical applications.  The blood of horseshoe crabs is harvested for this purpose.

H arvesting horseshoe crab blood involves collecting and bleeding the animals, and then releasing them back into the sea.

Horseshoe crabs donating their blue blood to create LAL for endotoxin testing.

Hardy Diagnostics now offers Associates of Cape Cod, Inc., Pyrosate Test Kits. These kits test for endotoxin presence using the Limulus Amaebocyte Lysate (LAL) gel clot method. When used according to the Limit Test and Quantitative Test procedures, these kits are fully compatible with United States Pharmacopeia (USP) Bacterial Endotoxin Test (BET) guidelines. Kits are sold in three sensitivity levels, 0.03 EU/mL, 0.125 EU/mL, and 0.25 EU/mL. Each sensitivity level is available in kits of 10 or 30 tests. Endotoxin-free transfer pipettes and reagent grade water are also available for use with these kits, if necessary.

The Pyrosate Test Kit is easy to use, rapid, and specific for endotoxin. Results are read simply by inverting the sample test tube. If a gel clot forms, the test is positive for endotoxin at or above the listed concentration.  Please refer to the product's Instructions for Use (IFU) for a more detailed description of the procedure and analysis of results.

To see the complete line of products, click HERE . If you have any questions regarding this new product line, please contact your Hardy Diagnostics Sales Representative or call Technical Services at 1-800-266-2222, option 2.

by Raquel Kahler