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How do you calculate bacterial endotoxin limits?

How do you calculate bacterial endotoxin limits

Endotoxins are large molecules composed of lipid & polysaccharides, which are found in the outer membrane of gram-negative bacteria. Lipopolysaccharides (LPS) greatly contributes to the structural integrity of the bacteria and is a major component of the protecting membrane from chemical attacks. Also, Bacterial endotoxin testing is necessary to prevent the patient from contracting a number of diseases.

Source – The primary source of endotoxin are gram-negative bacteria, some bacterial products, or debris. So, endotoxins are widely present in the environment, including dust, animal waste, foods, and other materials generated from or exposed to gram-negative bacterial products. Hence people involved in occupations like intensive livestock and/or agricultural operations get frequently exposed to higher levels of endotoxin.

Due to its prevalence in the environment, endotoxin has an endogenous presence in sites such as the airway and the gastrointestinal tract. They serve as bioaerosol through occupational exposure; they lead to the development & worsening of airway disease. Any additional injury that is mechanical or pathogen-induced (e.g., virus) allows for secondary bacterial invasion and thus lead to endotoxin presentation in living tissues or blood, hence initiating a robust immune response.

Physiology –Endotoxin being one of the most important bacterial components, is contributing to the inflammatory process. Its levels correlate directly with the severity of the meningococcal disease, sepsis, and release of inflammatory mediators.  It regulates TNF – alpha, coagulation pathways, altered gut permeability, etc. The anti endotoxin strategies involved in the management of sepsis neutralize endotoxin, enhance endotoxin clearance, or inhibit the endotoxin interaction with its receptors.

Properties – The first activity of an endotoxin being recognized is pyrogenicity, where they represent pyrogens or fever-inducing agents. There is a wide spectrum of biological activities that are demonstrated in vivo and in vitro. Among them, pyrogenicity, leukocytosis, induction of the local, generalized Schwartzman reaction, Platelet aggregation and lethal shock are some examples of classical endotoxic activities. Although most endotoxins are named after their toxic properties, they exhibit a good number of beneficial activities too. They induce nonspecific resistance to different infectious agents and to their own toxic effect, mitogenic stimulation, antibody synthesis, etc. Also you might have a lot of bacteria in your teeth. Inorder to clean it use the best service in australia that is dentist hervey bay dental clinic.

Testing / Safety check – Endotoxins tend to pose a significant risk to patients when left undetected. These high levels of endotoxins can enter into the bloodstream through a medical device & cause adverse reactions such as hemorrhagic shock, diarrhea, meningitis, fever, altered resistance to bacterial infection, a rapid drop in blood pressure, and other several biological effects.

Hence any pharmaceutical product that enters the body, including parenteral medicines and injectable devices, should be tested for the presence of endotoxins before being released into the market. These quality control (QC) tests must comply with regulatory requirements, and there are a variety of methods used. A few of them are Rabbit Pyrogen Test (RPT), Monocyte Activation Test (MAT), Limulus Amebocyte Lysate (LAL), etc.

Limulus Amoebocyte Lysate (LAL) –  Limulus Amoebocyte Lysate (LAL) assay is a fundamental safety requirement carried out to quantify this gram-negative bacteria within the cell wall.  Being more sensitive & specific, it tests medical devices/ agents meeting cerebrospinal fluid or the cardiovascular system to determine the presence of bacterial endotoxins. It uses a LAL reagent composed of specialized blood cells (amoebocytes) of Atlantic horseshoe crabs (Limulus polyphemus).

Amoebocytes form the crab’s primitive immune system and defensively clot when they encounter endotoxins (and other pathogens). The LAL reagent makes use of this clotting response to produce a BET assay that is more reliable for detection. Any product that comes in direct or indirect contact with intravascular, intra lymphatic, intrathecal and/or intraocular systems must undergo bacterial endotoxin testing (LAL) to confirm that the product’s endotoxin content is below the allowable release limit of 0.5 EU/mL.

Bacterial Endotoxin limits – Endotoxin’s limit for a medical component depends on the intended use of the device and what the component contacts with, for example, blood, the cardiovascular system, cerebrospinal fluid, intrathecal routes of administration, some permanently implanted devices or other implanted subcutaneously. It also depends on the process of preparing an extract for testing and it may vary from device to device. Some medical devices can be flushed, some may have to be immersed, while others may need disassembly or as per special standards.

Control Standard Endotoxins (CSEs) are an acceptable source of endotoxin preparations and the FDA encourages the continued use of CSEs that are suitably calibrated to the international endotoxins reference standard.

Endotoxin validation testing gives a more robust assessment of the test method, where inhibition and enhancement testing are performed simultaneously using multiple dilutions. These results are then assessed to see which method generates the closest results to the ideal recovery percentage and sample pH. The test must be repeated when there is any condition that has an influence on test results.

 

 

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