Limulus amebocyte lysate
Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the Atlantic horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria. This reaction is the basis of the LAL test, which is widely used for the detection and quantification of bacterial endotoxins.
Fred Bang reported in 1956 that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab to turn into a semi-solid mass. It was later recognized that the animal's blood cells, mobile cells called amoebocytes, contain granules with a clotting factor known as coagulogen; this is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation is thought to contain bacterial infections in the animal's semi-closed circulatory system.
In 1977 the U.S. Food and Drug Administration (FDA) approved LAL for testing drugs, products and devices that come in contact with the blood. Prior to that date, a much slower and more expensive test on rabbits had been used for this purpose.
Horseshoe crabs are collected and blood is removed from the horseshoe crab's pericardium; the crabs are then returned to the water. LAL manufacturers have measured mortality rates of 3% in bled crabs; however, recent studies indicate that this number may be closer to 15% or even 30%. The blood cells are separated from the serum using centrifugation and are then placed in distilled water, which causes them to swell and burst ("lyse"). This releases the chemicals from the inside of the cell (the "lysate"), which is then purified and freeze-dried. To test a sample for endotoxins, it is mixed with lysate and water; endotoxins are present if coagulation occurs.
The LAL test
There are three basic methodologies: gel-clot, turbidimetric, and chromogenic. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products.
The LAL cascade is also triggered by (1,3)-β-D-glucan. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns", or PAMPs, substances which elicit inflammatory responses in mammals.
Overcoming inhibition and enhancement
One of the most time consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement. Agents such as EDTA and heparin are known to affect the assay if they are present in sufficient concentrations. All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless the sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test. Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices. Proper endotoxin recovery must be proven before LAL can be used to release product.
Since 2003, a synthetic substitute for the LAL test has been commercially available. It is based on the Limulus clotting factor C protein, produced by genetically modified insect gut cells. The adoption of this test was slow, which began to change in 2016 when the European Pharmacopeia listed this test as an accepted bacterial-toxin test.
- Zhang, Sarah (2018-05-09). "The Last Days of the Blue-Blood Harvest". The Atlantic. Retrieved 2018-05-15.
- "Crash: A Tale of Two Species - The Benefits of Blue Blood". PBS. 10 June 2008.
- A.S. Leschen; S.J. Correia (February 25, 2010). "Mortality in female horseshoe crabs (Limulus polyphemus) from biomedical bleeding and handling: implications for fisheries management" (PDF). Archived from the original (PDF) on 2010-06-14.
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- Interference with the LAL Test and How to Address It, LAL Update, October 2005
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