Testing medical disposables using the Limulus Amoebocyte Lysate (LAL) test
The discovery of the horseshoe crab's most significant biological role in recent medicine was made by Frederick Bang in the early 1950's. Bang discovered that the horseshoe crab's blood cells, called amoebocytes, contain a clotting agent that attaches to dangerous endotoxins produced by gram negative bacteria. The test was accepted by the United States Food and Drug Administration (FDA) in 1983 as a standard test for endotoxins. In 1987, the FDA established guidelines for LAL testing of pharmaceuticals and medical devices.
During the early days of the pharmaceutical industry it was noticed that some solutions when injected into the bloodstream induced fevers. Investigations found that almost all of these fevers were associated with a group of contaminants termed pyrogens. These were classified as either exogenous or endogenous pyrogens. Exogenous pyrogens are fever causing materials found in the environment, of these endotoxins are the most researched and are lipopolysacchrides (LPS), found in the outer membrane of the cell wall of Gram -ve microorganisms, they are heat stable and can cause severe patient reactions when present in parenterals or medical devices. Endotoxin toxicity is not dependent upon a living cell, heat sterilization or other chemical/physical processes are ineffective control measures as killing the cells actually releases 'free' endotoxin from the cell wall.
As all mammals can be affected by endotoxins (although sensitivity levels vary) one of the first tests used to determine if endotoxins were present, was the Rabbit Pyrogen Test (RPT). A rabbit was inoculated with the test substance and then monitored it to see if a fever was induced. This test however does not give a quantitative result, is time consuming and is not suitable for products that may in themselves adversely affect the animal. The most commonly used approach now is a Limulus Amoebocyte Lysate or LAL test. The LAL test focuses in particluar on 2-keto-3-deoxyoctonoic acid and it is this which is used as an indicator in the majority of endotoxin assays. LAL is a reagent derived from the blood cells of the horseshoe crab, unlike a mammal the crab does not have a developed immune system, however the LAL component in its blood will bind to and inactivate endotoxins, in the crab the resulting clot also forms a protective barrier against bacterial infection. At its simplest the LAL test consists of adding LAL reagent to the sample in a test tube, incubating at 37°C for 1 hour. The tube is then gently inverted if a gel or clot has formed then a positive result is recorded.
The Gel-clot is the simplest and most widely used, it is also fully described by most pharmacopeias. The method can be qualitative or semi-quantitative when comparing a sample against a dilution series of an endotoxin standard. Can be used to provide a pass/fail for a certain limit and is best used with low sample numbers. The method may be performed manually with little or no requirement for instrumentation. Because the potency of an endotoxin to cause pyrogenic reaction will vary according to the nature of the toxin, the FDA developed Endotoxin Units (EU) for result comparisons.
