Limulus amebocyte lysate (LAL) is an aqueous extract of

motile Motility is the ability of an organism to move independently using metabolic energy. This biological concept encompasses movement at various levels, from whole organisms to cells and subcellular components. Motility is observed in animals, mi ...

blood cell A blood cell (also called a hematopoietic cell, hemocyte, or hematocyte) is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), ...

s ( amebocytes) from the

Atlantic horseshoe crab The Atlantic horseshoe crab (''Limulus polyphemus''), also known as the American horseshoe crab, is a species of horseshoe crab, a kind of marine biology, marine and Brackish water, brackish chelicerate arthropod. It is found in the Gulf of Mexic ...

''Limulus polyphemus''. LAL reacts with bacterial endotoxins such as

lipopolysaccharide Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...

s (LPS), which are components of the

bacterial capsule The bacterial capsule is a large structure common to many bacteria. It is a polysaccharide layer that lies outside the cell envelope, and is thus deemed part of the outer envelope of a bacterial cell. It is a well-organized layer, not easily wash ...

, the outermost membrane of

cell envelope The cell envelope comprises the inner cell membrane and the cell wall of a bacterium. In Gram-negative bacteria an bacterial outer membrane, outer membrane is also included. This envelope is not present in the Mollicutes where the cell wall is abse ...

gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...

. This reaction is the basis of the LAL test, which is widely used for the detection and quantification of bacterial

endotoxin Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...

s. In Asia, a similar Tachypleus amebocyte lysate (TAL) test based on the local horseshoe crabs '' Tachypleus gigas'' or '' Tachypleus tridentatus'' is occasionally used instead. The recombinant factor C (rFC) assay is a replacement of LAL and TAL based on a similar reaction.

Background

The American medical researcher

Fred Bang Frederik Barry Bang (1916–1981) was an American medical researcher who developed the Limulus amebocyte lysate (LAL) test for bacterial endotoxins. He was influential in applying marine biology to medical research, especially immunology. Biogra ...

reported in 1956 that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab to turn into a gel, a type of semi-solid mass. It was later recognized that the animal's blood cells, mobile cells called amebocytes, contain granules with a clotting factor known as coagulogen; this is released outside the cell when bacterial endotoxins are encountered. After

coagulation Coagulation, also known as clotting, is the process by which blood changes from a liquid to a gel, forming a thrombus, blood clot. It results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The process of co ...

and subsequent gelling, the resulting gel is thought to contain bacterial infections in the animal's semi-closed

circulatory system In vertebrates, the circulatory system is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the body. It includes the cardiovascular system, or vascular system, that consists of the heart ...

. Modern analysis of the lysate has led to understanding of this system of cascade, with multiple enzymes working in sequence to produce the gel. The entry point of endotoxin-induced clotting is Limulus clotting factor C. In 1977 the

U.S. Food and Drug Administration The United States Food and Drug Administration (FDA or US FDA) is a federal agency of the Department of Health and Human Services. The FDA is responsible for protecting and promoting public health through the control and supervision of food ...

(FDA) approved LAL for testing drugs, products and devices that come in contact with 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 pericardium (: pericardia), also called pericardial sac, is a double-walled sac containing the heart and the roots of the great vessels. It has two layers, an outer layer made of strong inelastic connective tissue (fibrous pericardium), ...

; some crabs are then returned to the water, while others are sold to be eaten or used as bait. Companies extracting LAL from horseshoe crabs stated before 2008 that mortality rates were below 3%. A 2009 Massachusetts Division of Marine Fisheries study stated that earlier studies found 5 to 15% mortality for males and one estimate of 29% for females. The study itself found 22% for females returned immediately to the water, and 30% for females kept overnight to represent commercial practice. The blood cells are separated from the serum using

centrifugation Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate ...

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 Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product and lowering pressure, thereby removing the ice by sublimation. This is in contrast to dehydration by m ...

. 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 In chemistry, the term chromogen refers to a colourless (or faintly coloured) chemical compound that can be converted by chemical reaction into a compound which can be described as "coloured" (a chromophore). There is no universally agreed definiti ...

. The primary application for LAL is the testing of

parenteral In pharmacology and toxicology, a route of administration is the way by which a drug, fluid, poison, or other substance is taken into the body. Routes of administration are generally classified by the location at which the substance is applied. ...

pharmaceuticals and medical devices that contact

blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood is com ...

cerebrospinal fluid Cerebrospinal fluid (CSF) is a clear, colorless Extracellular fluid#Transcellular fluid, transcellular body fluid found within the meninges, meningeal tissue that surrounds the vertebrate brain and spinal cord, and in the ventricular system, ven ...

. 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 Cascade, or Cascading may refer to: Science and technology Science * Air shower (physics), a cascade (particle shower) of subatomic particles and ionized nuclei ** Particle shower, a cascade of secondary particles produced as the result of a high ...

is also triggered by (1,3)-β-D-glucan, via a different Factor G. Both bacterial endotoxins and (1,3)-β-D-glucan are considered

pathogen-associated molecular pattern Pathogen-associated molecular patterns (PAMPs) are small molecular motifs conserved within a class of microbes, but not present in the host. They are recognized by toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) in both p ...

s, or PAMPs, substances which elicit inflammatory responses in

mammal A mammal () is a vertebrate animal of the Class (biology), class Mammalia (). Mammals are characterised by the presence of milk-producing mammary glands for feeding their young, a broad neocortex region of the brain, fur or hair, and three ...

Overcoming inhibition and enhancement

One of the most time-consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition that 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.

Alternatives

Recombinant factor C assay

The LAL test is a major source of

animal product An animal product is any material derived from the body of a non-human animal or their excretions. Examples are meat, fat, blood, milk, eggs, honey, and lesser known products, such as isinglass, rennet, and cochineal. The word animals inc ...

dependence in the biomedical industry, and a challenge to the Three Rs of science in relation to the use of animals in testing. With reports of higher-than anticipated mortality rates it has been considered more ethical to devise alternatives to the test. Since 2003, a recombinant protein substitute for use in the LAL test has been commercially available. Named the recombinant factor C (rFC) assay, it is based on the same Limulus clotting factor C protein, but produced by genetically modified insect cells (the specific factor C sequence used does not necessarily come from the Atlantic horseshoe crab). Instead of emulating the whole clotting pathway, rFC tests let factor C cleave a synthetic fluorogenic substrate, so that the sample lights up when endotoxin activates the factor. Since it does not contain factor G, (1,3)-β-D-glucan will not cause

false-positive A false positive is an error in binary classification in which a test result incorrectly indicates the presence of a condition (such as a disease when the disease is not present), while a false negative is the opposite error, where the test resu ...

s. As of 2018, available evidence shows that the rFC test is no worse than the LAL test. The adoption of the rFC test was slow, which began to change in 2012 when the US

FDA The United States Food and Drug Administration (FDA or US FDA) is a federal agency of the Department of Health and Human Services. The FDA is responsible for protecting and promoting public health through the control and supervision of food ...

and the European health ministry acknowledged it as an accepted alternative. Its lack of mention in Pharmacopeias remained an issue, as there was no good standard for running the test in production. In 2016, it was added to the

European Pharmacopoeia The ''European Pharmacopoeia'' (''Pharmacopoeia Europaea'', ''Ph. Eur.'') is a major regional pharmacopoeia which provides common quality standards throughout the pharmaceutical industry in Europe to control the quality of Pharmaceutical drug, ...

. A patent on rFC also limited adoption until its expiration in 2018. On 1 June 2020, the

United States Pharmacopeia The ''United States Pharmacopeia'' (''USP'') is a pharmacopeia (compendium of drug information) for the United States published annually by the over 200-year old United States Pharmacopeial Convention (usually also called the USP), a nonprofi ...

(USP) decided to cancel the proposal to include recombinant technology for endotoxin testing in chapter 85, ''Bacterial Endotoxins'', and start the development of a separate chapter that expands on the use, validation, and comparability of endotoxin tests based on recombinantly derived reagents. A separate guidance-only chapter 1085.1 was proposed by the USP, though comments and feedback published on 11 December 2020 show that pharmaceutical companies and the FDA do not support this chapter, and request for compendial status.

Monocyte activation test

The monocyte activation test (MAT) is another proposed method to test for endotoxins based on

monocyte Monocytes are a type of leukocyte or white blood cell. They are the largest type of leukocyte in blood and can differentiate into macrophages and monocyte-derived dendritic cells. As a part of the vertebrate innate immune system monocytes also ...

s in

human blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood is compo ...

. It measures the release of

cytokines Cytokines () are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B cell, B lymphocytes, T cell, T lymphocytes ...

from these due to the presence of pyrogens, basically mirroring the process by which these toxins cause fever in humans (and rabbits, as in the original pyrogen test). A protocol for the MAT test, using cultured cells, is described in the European Pharmacopoeia. A recent study employing genetically engineered monocytes was able to significantly enhance the sensitivity of monocyte-based detection assays by bringing down the assay-completion time from more than 20 hours to 2–3 hours.

References

External links

horseshoecrab.org

{{Webarchive, url=https://web.archive.org/web/20100714005129/http://indradiagnostics.com/LAL.html , date=14 July 2010
1987 FDA LAL test guideline LAL Test Reagents
Chemical tests