Bacterial Endotoxin Testing, also referred to as BET, is a necessary safety requirement in pharmaceuticals and biomedical industries. When bacterial endotoxins enter a person’s bloodstream, it can cause severe symptoms based on the concentrations.
For this reason, all injectable devices or parenteral medicines must undergo tests to identify the presence of endotoxins before these are released into the market. These QC tests must conform to the requirements of regulatory bodies like the FDA.
WHAT IS BACTERIAL ENDOTOXIN?
It is a kind of pyrogen, which are agents of fever. It is present in the cell wall of gram-negative bacteria.
WHAT ARE ITS IMPLICATIONS?
Bacterial endotoxin is one of the lethally dangerous and commonly occurring pyrogenic (fever-causing) agents due to the following reasons:
- Its ubiquitous nature. These contaminants are capable of multiplying in clear water.
- It is potently toxic.
- It is relatively stable even in extreme conditions.
- It widely occurs during manufacturing processes.
Depending upon the concentration, when these enter the bloodstream, or even spinal fluid, it can cause septic shock, fever, organ failure, and death in some cases. Due to these drastic effects, all injectable or implantable devices and pharmaceutical products that come into contact with blood are tested for the presence of endotoxins. It is to ensure the safety of patients.
NEED FOR TESTING:
Due to possibilities of drastic consequences, or death, all injectable devices and products are made to undergo tests for endotoxins with bacterial endotoxin testing. The most commonly used BET test uses LAL or Limulus amebocyte Lysate to check for endotoxins.
Reliable, efficient, and quick testing is vital in biomedical industries. All implants or injectable devices must undergo this test to get labeled as sterile or pyrogen-free. This test not only protects the patients from the harmful effects but also complies with the regulations. LAL assay is a widely used and well-established testing procedure to identify endotoxins.
TYPES OF TESTS:
These tests reveal simple results in the form of yes or no answers regarding the presence of endotoxins.
LAL test (Simple): This does not need an incubating reader or software. It visually inspects the gel formation.
It not just reveals the presence of these endotoxins but also indicates the amount present.
LAL Assay: Chromogenic
It does an endpoint measurement of certain color developments to allow quantitative results of the concentration of endotoxins in a given sample.
LAL Assay: Turbidimetric
It does a kinetic measurement of the turbidity development. It is a comparatively cost-effective way to test large samples.
Recombinant factor C:
It is a non-animal substitute to LAL tests. It uses recombinantly produced factor C and is the first constituent in a clotting cascade of a horseshoe crab. It does not depend on its blood as the testing reagent of the source.
WHY IS TESTING IMPORTANT:
It is vital because these endotoxins can get access even from a simple contact with parenteral devices, medicines, and equipment that have been contaminated. It is essential when taking care of older people as they are more susceptible to its effects.
Vulnerable people include elderly patients and patients undergoing treatment, Intensive Care. Take, for example, SIDS- Sudden Infant Death Syndrome. It is associated with endotoxins being present in the bloodstream in several cases.
When injectable devices were introduced, there was no established method to test for pyrogenic contaminants. If a person got injected, there was a high chance that that person would get a fever. It was usually called ‘injection fever.’
METHODS OF TESTING:
There are various methods to detect pyrogens like bacterial endotoxins. The RPT was a traditional approach that used a qualitative approach. The principle behind this test was that fever-inducing contaminants induced temperature variation in rabbits.
This method is, however, a general test that is not specific to bacterial endotoxin. The MAT is a more recent approach, developed in the 1900s. After that, numerous testing methods have evolved to detect pyrogens efficiently.
LAL Assay :
The Limulus Amebocyte Lysate has replaced the RPT due to its efficacy. It is comparatively more sensitive and a specific testing method for BET. This test uses a LAL reagent made with amebocytes (specific blood cells) of Atlantic horseshoes crabs.
These cells have a primitive form of the immune system. They defensively clot as soon as they come into contact with endotoxins or other pathogens. The reagent uses this clotting phenomenon to offer a BET assay that is both reliable and specific.
There is an acceptable level for the presence of endotoxins which is 0.5 units per ml. If the level exceeds this quantity, manufacturers of such devices might face trouble. BET plays a vital role in ensuring the quality of medical devices and equipment.