Horseshoe Crab is used by Costa Rican laboratories to evaluate injectables.

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The coagulation of a reagent produced from the blood of the horseshoe crab allows the Laboratory of Standards and Quality of Medicines (LNCM) to confirm that the injectables used in the Costa Rican Social Security Fund (CCSS) are free of bacteria dangerous to health from the patients.

A component of horseshoe crab blood is used as an input because it reacts to the presence of what the outer walls of bacteria are made of (bacterial endotoxins), causing the liquid medium to clot and reveal its contamination, says Dr. Mario André Oreamuno Ávila, in charge of the Bioanalysis Unit of the LNCM.

The coagulation of a reagent produced from the blood of the horseshoe crab allows the Laboratory of Standards and Quality of Medicines (LNCM) to confirm that the injectables used in the Costa Rican Social Security Fund (CCSS) are free of bacteria dangerous to health from the patients.

A component of horseshoe crab blood is used as an input because it reacts to the presence of what the outer walls of bacteria are made of (bacterial endotoxins), causing the liquid medium to clot and reveal its contamination, says Dr. Mario André Oreamuno Ávila , in charge of the Bioanalysis Unit of the LNCM.

The harmful effects of bacteria walls

This method of analysis by coagulation represented a great advance in the verification of the quality of medicines, since bacterial endotoxins are related to mild, moderate and serious damage to humans.

Endotoxins are the structures on the outer cell walls that bacteria use to maintain the integrity of their protective membrane and ensure their survival. These structures are formed especially by a type of fats and sugars with harmful effects on health.

Among the best known bacteria that could generate endotoxins are Eschericia coli, Salmonella, Shigella and Pseudomonas, among others.

When entering the bloodstream, the body’s defenses generate fever, that is, an increase in body temperature, and an inflammatory response, dilation of blood vessels, bleeding, shock and, in extreme cases, can even cause death.

From rabbits to crabs in the safety of injectables

Internationally, as well as in the LNCM, there was a time when rabbits were used in tests to detect the presence or absence of endotoxins in drugs.

The test consisted of injecting the product sample into the ear of a rabbit and waiting for the reaction in body temperature: if an endotoxin was present in the injected substance it caused the animal’s body temperature to rise above normal.

This was efficient because rabbits develop fever responses to endotoxins in a similar way to humans, which is why they were used as a model in screening tests.

For more than 30 years the test in rabbits was the method of choice in the official pharmacopoeias.

However, for practical reasons, currently the main international regulatory authorities require the control of endotoxins in pharmaceutical products through laboratory studies, which is an in vitro method that manages to detect the presence of bacterial endotoxins with high sensitivity.

The protagonist in this new stage is the horseshoe crab, also called a living fossil, as it is one of the oldest living beings that exist, since they have inhabited the planet before the dinosaurs.

Its entry as a star among pharmacological materials began in the 1950s, when North American scientists discovered that the blue-colored blood of this crab coagulated when it came into contact with live bacteria such as Escherichia coli and Salmonella, as well as with bacterial toxins, forming a kind of gelatin jail that immobilized bacteria and sealed their way to the rest of his body.

Based on this principle, scientists developed a purified extract of crab blood cells that reacts chemically when in the presence of bacterial endotoxins and that forever changed safety studies on injectable drugs.

Currently the test consists of diluting the drugs to a maximum allowed level and putting them in contact with the purified extract, which reacts by generating a perfectly formed clot in case of detecting endotoxins in the product, after an incubation period with controlled temperature and time.

To carry out this test, the laboratory has facilities with strict environmental controls and specialized equipment (depyrogenizing oven, laminar flow cabinets, sample incubators that maintain controlled temperature, depyrogenized glassware) as well as highly qualified technical personnel.

Every medicine receives quality control before it is delivered

The Medicines Quality and Standards Laboratory -ascribed to the Caja’s Logistics Management- is the entity in charge of verifying and monitoring the quality of the pharmaceutical products acquired and produced by the Institution, prior to their dispatch in the supply chain and during its useful life in the CCSS.

One of the many studies that are part of the set of analyzes carried out by this Laboratory is the test for the detection of bacterial endotoxins. This analysis is performed by clotting a reagent produced from the blood of a crab called Limulus polyphemus.

This procedure is accredited under the ISO 17025: 2017 standard.

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